Pathophysiology of Exercise Intolerance in Breast Cancer Survivors Treated with

Anthracycline Chemotherapy

Rhys I. Beaudry

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at The

University of Texas at Arlington

December, 2019

Arlington, Texas

Committee:

Dr. Mark J. Haykowsky, Supervising Professor

Dr. Michael D. Nelson

Dr. R. Matthew Brothers

Dr. Satyam Sarma

ii

Abstract

Pathophysiology of Exercise Intolerance in Breast Cancer Survivors Treated with

Anthracycline Chemotherapy

Rhys I. Beaudry, Ph. D

The University of Texas at Arlington

2019

Supervising Professor: Mark J. Haykowsky

Anthracyclines emerged as frontline breast cancer adjuvant therapy in the late 1960’s. Within half a

decade of their clinical adoption, dose-limiting cardiotoxicity was recognized and cumulative dose limits

were established to acceptably balance anti-tumor and cardiotoxic properties. Despite these longstanding

dose limits, anthracycline treated breast cancer (BC) survivors have advanced biological aging, as

evidenced by a marked impairment in peak oxygen uptake (VO2 peak). Given that the pathophysiology of

reduced VO2 peak in BC is poorly understood, the aim of this dissertation was to: 1) develop a non-invasive

imaging technique to investigate cardiac function under exercise stress; 2) investigate the cardiac limits to

exercise in BC; and 3) investigate muscle blood flow, oxygen use, and bioenergetics as components of

exercise intolerance in BC.

In study 1 (Chapter 2) an exercise cardiac magnetic resonance imaging (cMRI) technique was developed

using commercially available hardware and in-product image sequences. Rest and exercising cardiac

volumes were measured in eight young healthy subjects (mean age: 25 years) and a meta-analysis was

performed to demonstrate consistency of results, thus demonstrating feasibility and establishing the

normative cardiac response to supine exercise cMRI. From rest to exercise, cardiac output increased by an

increase in heart rate and stroke volume, through preserved end-diastolic volume and reduced end-systolic

iii

volume. In study 2 (Chapter 3), using exercise cMRI and cardiopulmonary exercise testing, cardiac

function and VO2 peak were measured in 29 BC patients (mean age: 48 years) prior to receiving

anthracycline based chemotherapy and in 10 age- and sex-matched healthy controls. On average, BC

patients had 20% lower VO2 peak (l/min or ml/kg/min) than healthy controls, which was related to lower

peak exercise cardiac output. Importantly, decrements in peak cardiac output were apparent prior to

anthracycline administration. In study 3 (Chapter 4), using femoral artery Doppler ultrasound, leg blood

flow was measured during single-leg knee extension (SLKE) in 14 BC survivors (mean age: 61 years,

mean time post anthracycline therapy: 12 years) and 9 age- and sex-matched controls. Peak SLKE power

output, heart rate and blood pressure were comparable between groups. Leg (femoral artery) blood flow

was measured during 25, 50 and 75% of peak SLKE, and was not significantly different between groups

at rest or during submaximal exercise. However, estimated quadriceps muscle mass was reduced in BC

survivors, despite normal leg blood flow and conductance. In study 4 (Chapter 5), using MRI, lower leg

(superficial femoral vein) blood flow, VO2 and bioenergetics were measured during plantar flexion

exercise in 16 BC survivors (mean age: 56 years, mean time post anthracycline therapy: 13 months) and

16 age-, sex- and body mass index matched controls. Muscle oxidative capacity was not impaired, nor

was muscle blood flow or oxygen extraction. However, BC survivors tended to have abnormal leg

composition (increased fat and reduced muscle) that contributed to reduced VO2 peak. Taken together, the

data herein demonstrate a strong non-cardiac component to exercise intolerance in anthracycline treated

BC survivors, similar to that found in sex- and age-matched controls. Future research building upon non-

cardiac interventions for prevention of anthracycline related reductions in VO2 peak are needed to reduce

cardiovascular risk in BC survivors.

iv

Copyright by

Rhys I. Beaudry, 2019

v

Acknowledgements

This work was made possible by the mentorship and academic and financial support of my supervising

professor, Dr. Mark Haykowsky, as well as committee members Drs. Satyam Sarma, Michael D. Nelson

and R. Matthew Brothers.

vi

Dedication

To all the individuals that have provided invaluable support along the way- friends, family, mentors,

committee members, Texas Moms, thank you. Your guidance within and outside of academia has been

instrumental in my development, and I hope will lead to lifelong relationships.

vii

Table of Contents Abstract ..................................................................................................................................................... ii

Acknowledgements ................................................................................................................................... v

Dedication ................................................................................................................................................ vi

Table of Contents ........................................................................................................................................ vii

List of Figures ........................................................................................................................................... ix

List of Tables ............................................................................................................................................ ix

..................................................................................................................................................... 10

1.1 Pathophysiology of Exercise Intolerance in Breast Cancer: Review of the literature. ..................... 10

1.1 a) Introduction: ............................................................................................................................. 11

1.1 b) Anthracycline Cardiotoxicity: Evolution to the present state of knowledge: ........................... 12

1.1 c) Exercise Intolerance in Breast Cancer: ...................................................................................... 15

1.1 d) Statement of the problem: ....................................................................................................... 24

1.1 e) References ................................................................................................................................ 26

..................................................................................................................................................... 29

2.1 Exercise Cardiac Magnetic Resonance Imaging: A feasibility study and Meta-Analysis. .................. 29

2.1 a) Abstract ..................................................................................................................................... 30

2.1 b) New & Noteworthy: .................................................................................................................. 31

2.1 c) Introduction .............................................................................................................................. 32

2.1 d) Methods .................................................................................................................................... 33

2.1 e) Results ....................................................................................................................................... 37

2.1 f) Discussion .................................................................................................................................. 39

2.1 g) Conclusion ................................................................................................................................. 42

2.1 h) References ................................................................................................................................ 43

2.1 i) Figure Legends: .......................................................................................................................... 48

2.1 j) Tables ......................................................................................................................................... 49

2.1 k) Figures ....................................................................................................................................... 52

..................................................................................................................................................... 54

3.1 Determinants of Exercise Intolerance in Breast Cancer Patients Prior to Anthracycline

Chemotherapy ........................................................................................................................................ 54

3.1 a) Abstract ..................................................................................................................................... 55

3.1 b) Introduction .............................................................................................................................. 56

3.1 c) Methods .................................................................................................................................... 57

viii

3.1 d) Results ....................................................................................................................................... 60

3.1 e) Discussion.................................................................................................................................. 62

3.1 f) Conclusion.................................................................................................................................. 65

3.1 g) References................................................................................................................................. 66

3.1 h) Figure Legends .......................................................................................................................... 72

3.1 i) Tables ......................................................................................................................................... 73

3.1 j) Figures ........................................................................................................................................ 75

..................................................................................................................................................... 79

4.1 Leg blood flow is preserved during small muscle mass exercise in long-term survivors of

anthracycline treated breast cancer. ...................................................................................................... 79

4.1 a) Abstract: .................................................................................................................................... 80

4.1 b) Introduction .............................................................................................................................. 81

4.1 c) Methods: ................................................................................................................................... 82

4.1 d) Results: ...................................................................................................................................... 85

4.1 e) Discussion.................................................................................................................................. 86

4.1 f) References ................................................................................................................................. 90

4.1 g) Figure Legends: ......................................................................................................................... 92

4.1 h) Tables: ....................................................................................................................................... 93

4.1 i) Figures: ....................................................................................................................................... 96

..................................................................................................................................................... 99

5.1 Exercise intolerance in anthracycline-treated breast cancer survivors: the role of skeletal muscle

bioenergetics, oxygenation and composition. ........................................................................................ 99

5.1 a) Abstract ................................................................................................................................... 100

5.1 b) Implications for Practice: ........................................................................................................ 101

5.1 c) Introduction ............................................................................................................................ 102

5.1 d) Methods .................................................................................................................................. 103

5.1 e) MR Imaging and 31P Magnetic Resonance Spectroscopy ....................................................... 104

5.1 f) Results ...................................................................................................................................... 108

5.1 g) Conclusion ............................................................................................................................... 115

5.1 h) References .............................................................................................................................. 116

5.1 a) Figure Legends: ....................................................................................................................... 119

5.1 b) Figures: .................................................................................................................................... 120

5.1 c) Tables: ..................................................................................................................................... 123

................................................................................................................................................... 128

ix

6.1 Conclusion ....................................................................................................................................... 128

6.1 a) References .............................................................................................................................. 133

................................................................................................................................................... 134

7.1 Appendix ......................................................................................................................................... 134

7.1 a) Chapter 3 Data Supplement .................................................................................................... 135

List of Figures Figure 1: cMRI Images and Results ............................................................................................................. 52

Figure 2: Meta-Analysis Results .................................................................................................................. 53

Figure 3: Left Ventricular Volumes; rest to peak exercise .......................................................................... 75

Figure 4: Right Ventricular Volumes; rest to peak exercise ........................................................................ 76

Figure 5: Heart Rate and Cardiac Index; rest to peak exercise ................................................................... 77

Figure 6: Peak Cardiac Output and Peak Oxygen Uptake ........................................................................... 78

Figure 7: Power output, leg blood flow, mean arterial pressure and leg conductance at rest and during

single leg knee extension ............................................................................................................................ 96

Figure 8: Femoral artery pressure-strain .................................................................................................... 97

Figure 9: Experimental set-up for plantarflexion exercise and image acquisition for determination of

lower-leg oxygen uptake ........................................................................................................................... 120

Figure 10: MRI body composition acquisition and analysis ...................................................................... 121

Figure 11: Relationship between IMF:SM ratio and whole body peak oxygen uptake ............................ 122

List of Tables Table 1: Metabolic and hemodynamic comparison between supine and upright exercise ....................... 49

Table 2: Characteristics of exercise cMRI studies reporting LV Volumes ................................................... 50

Table 3: Meta-Analysis effect size, significance and heterogeneity ........................................................... 51

Table 4: Participant Characteristics ............................................................................................................ 73

Table 5: Cardiopulmonary exercise performance during upright cycle exercise ....................................... 74

Table 6 Participant Characteristics ............................................................................................................. 93

Table 7 Maximal single leg knee extension parameters ............................................................................. 94

Table 8 Pearson's correlations between femoral artery blood flow and pressure-strain .......................... 95

Table 9 Participant characteristics ............................................................................................................ 123

Table 10 Cardiopulmonary exercise performance and lower leg muscle bioenergetics .......................... 124

Table 11 Body composition of the thigh, lower leg and abdomen ........................................................... 126

Table 12 Pearson's correlations between lower leg IMF:SM ratio and skeletal muscle bioenergetics ... 127

1.1 Pathophysiology of Exercise Intolerance in Breast Cancer: Review of the

literature.

11

1.1 a) Introduction:

Breast cancer (BC) is the most frequently diagnosed malignancy in women, and the

second leading cause of cancer mortality.1 Approximately 1 out of every 8 women will be

diagnosed with BC in their lifetime, amounting to nearly a quarter million new cases annually in

the United States.2 Over the past 4 decades, improvements in prevention, detection and treatment

have resulted in a nearly 40% BC mortality reduction.1 A direct implication of improved survival

is the rapidly expanding population of survivors, for which competing causes of morbidity and

mortality are growing concerns. Specifically, cardiovascular disease (CVD) is a leading cause of

death in BC survivorship.3 Most strikingly is the reported 35% increase (adjusted incidence rate

ratio) in risk of cardiomyopathy/heart failure (HF).4 This excess risk has traditionally been

attributed to cardiotoxic BC therapies including mediastinal chest irradiation and chemotherapy;

however, in 2007 this understanding evolved to the “Multiple Hit Hypothesis”, explaining excess

risk as a culmination of underlying CVD risk factors at diagnosis, direct cardiotoxic anti-cancer

therapy, and indirect effects of anti-cancer therapy (poor nutrition, decline in physical activity).5

Limited data were available to support the Multiple Hit Hypothesis when it was proposed;

however, over the last decade a wealth of new data has emerged from the physiologic to

epidemiologic level confirming and advancing the understanding of elevated CV mortality in BC

survivorship. The present paradigm explaining excess CV risk posits a combination of pre-

existing CVD risk factors, shared biologic pathways between CVD and BC, and treatment and

lifestyle toxicity result in elevated CV mortality.6

Herein, the focus will be anthracycline related cardiotoxicity and HF in BC.

Anthracycline class drugs were first introduced to oncology practice in the late 1960’s.7, 8 Their

12

robust antitumor efficacy resulted in widespread use and anthracycline based chemotherapy

regimens quickly became frontline therapy. Within 5 years of their clinical induction, dose

limiting cardiotoxicity was reported.9 Today, anthracyclines remain a treatment mainstay; anti-

tumor effects must carefully be balanced with risk of cardiotoxicity.

1.1 b) Anthracycline Cardiotoxicity: Evolution to the present state of knowledge:

In 1979 the first largescale study of anthracycline related HF was released.10 This study

consisted of retrospective analysis of 4018 anthracycline treated patients with histopathological

examination of HF cases, as well as post-mortem non-HF cases when available.10 Von Hoff et al.

found overall incidence of doxorubicin (the clinical standard anthracycline at the time) induced

HF was 2.2%; a conservative cumulative incidence given that many patients received low doses,

and only clinically diagnosed HF cases were counted. Heart failure occurred on average 33 days

after the last doxorubicin dose, and was lethal in 60% of patients. Remarkably, only 37% of HF

cases demonstrated histopathological evidence of doxorubicin cardiotoxicity, and an additional

8% of a cohort subset (n=347) showed post-mortem evidence of subclinical damage. These

investigators identified three factors associated with cardiotoxicity; first, they confirmed earlier

reports of cumulative dose as the primary risk factor. Second, dosing schedule appeared to

influence development of cardiotoxicity, with a trend for greater peak plasma concentration

eliciting cardiotoxicity. Third, increasing age was associated with cardiotoxicity however it was

unclear if the latter finding was related to age, or age associated declines in CV health (i.e.

functional status, cardiovascular comorbidities).

This study identified two critical research areas10. First, research was needed to advance

cardiotoxicity risk stratification, and improve early detection/monitoring and therapy for

13

cardiotoxicity. In the cohort, the leading cause of death was progressive malignant disease,

establishing the need for higher cumulative doses of doxorubicin in patients at low risk of

cardiotoxicity and HF. The authors recognized that their sample was drawn from clinical trials

prone to selection bias through exclusion of “sick” patients, thus limiting application of their

results. Underlying CV risk factors and low functional status were previously demonstrated as

relevant risk factors for cardiotoxicity, but were not statistically significant in the selected

retrospective analysis. As suggested by the authors, the use of developing methodologies

(cardiac biomarkers, ECG, echocardiography) for monitoring cardiac function and detecting

subclinical cardiotoxicity could aid in earlier and more effective intervention. More research was

needed to identify effective therapies for reversal of cardiotoxicity.

Second, Von Hoff et al.10 reported that 1) subclinical cardiotoxicity (post-mortem

evidence of cardiac damage without HF) was present in 8% of non-HF cases, and 2) the vast

majority of HF cases were not consistent with doxorubicin induced cardiac damage. This

curious phenomenon marks a discord between anthracycline mediated cardiotoxicity and

development HF, and raises the possibility of an indirect relationship between anthracyclines and

HF.

The present state of knowledge of anthracycline cardiotoxicity is based on the seminal

study by Cardinale et al. who demonstrated the feasibility of early detection and pharmaceutical

intervention to reverse cardiotoxicity.11 This prospective study was designed to elucidate the

prevalence and pathophysiology of anthracycline cardiotoxicity through serial monitoring using

state of the art echocardiography.11

At the time, the accepted model of anthracycline cardiotoxicity included 3 subtypes: 1)

acute, occurring after a single dose/course with clinical manifestation in less than 2 weeks, 2)

14

early-onset chronic, developing within 1 year of therapy and characterized by cardiac

hypokinesis, and 3) late-onset chronic, developing years to decades after therapy. Sparse data

were available to support the model, as the vast majority of studies were retrospective, and only

included patients who had developed clinically overt HF. As a result, it was unclear when

cardiotoxicity occurred in relation to the onset of clinical HF. To address this, Cardinale et al.11

prospectively enrolled 2625 consecutive patients scheduled for anthracycline based

chemotherapy and monitored patient’s quarterly using echocardiography. Cardiotoxicity was

defined as a decline in left ventricular ejection fraction (LVEF) by 10% or to a value below 50%

(biplane) and evidence based anti-HF medications were administered given evidence of

cardiotoxicity. Notably, this definition of cardiotoxicity is not synonymous with clinical

presentation of HF, but identifies subclinical cardiac dysfunction which may progress to clinical

cardiac dysfunction and overt HF.

Cardinale et al.11 reported the presence of cardiotoxicity in 9.7% of BC patients, similar

to findings by Von Hoff et al. more than 3 decades earlier.10 Ninety eight percent of cases

occurred within the first year (median 3.5 months) of therapy cessation, and when treated with

contemporary HF medication, 11% of patients had full recovery, 71% made partial recovery

(defined as an increase in LVEF to >50% or an increase in LVEF by 5 percentage points), while

18% showed no improvement.11 The authors argued for a singular pathophysiology of early

onset cardiotoxicity, which could insidiously decline until symptomatic detection of HF years

after incipient damage if left undetected and untreated. In spite of their compelling evidence that

98% of cases occurred within the first year, the median follow-up was only 5 years, with few

cases available beyond 10 years. Importantly, as in the general population, age is the greatest

risk factor for the development of CVD and HF with women diagnosed with BC who are >7

15

years having nearly 23-fold greater risk of developing HF relative to women diagnosed between

50 and 54 years of age.12

Detection of cardiotoxicity resulting in cardiac dysfunction was contingent upon changes

in resting cardiac function, as measured by echocardiography.11 Cardinale et al. provide

promising results with this clinically relevant and cost effective strategy, however, there remains

discord between resting cardiac measures and exercise intolerance- the hallmark feature of HF.

Cardiac stress imaging and more robust non-invasive imaging strategies could provide earlier

insight into functional declines and changes in cardiac tissue (i.e. fibrosis), opening the window

for potentially earlier and more effective intervention.

1.1 c) Exercise Intolerance in Breast Cancer:

The hallmark feature of HF is exercise intolerance, measured objectively as decreased

peak oxygen uptake (VO2 peak) during maximal exercise. VO2 peak is the strongest independent

predictor of CV, and all-cause mortality in healthy and clinical populations, and is related to

survival in BC.13-15 Across the survivorship continuum, VO2 peak is approximately 20% lower

relative to age and sex matched controls.13, 16-18 This decrement in cardiorespiratory fitness likely

contributes to elevated CVD risk as a 40 year-old BC survivor has been shown to have a VO2 peak

similar to that found in healthy sedentary women 20-30 years older.13 Currently, the mechanisms

underpinning impairments in VO2 peak are poorly understood.

In accordance with the Fick principal (VO2 = the product of cardiac output [Q] and

arterial-venous oxygen difference [CaO2 – CvO2]), the reduced in VO2 peak may be due to central

and/or peripheral, factors with the caveat that there is interdependency between flow and oxygen

extraction equilibration.

16

1.1 c) i: Cardiac Determinants of impaired VO2peak:

Cardiac output is the product of heart rate (HR) and stroke volume (SV), the difference

between end-diastolic (EDV) and end-systolic volume (ESV). Given the known cardiotoxic

nature of anthracycline chemotherapy, impaired peak Q may be an important contributor to

exercise intolerance in anthracycline treated BC survivors. To date, 3 previous investigations

have examined cardiac function in anthracycline treated BC survivors.

Jones et al. using impedance cardiography and expired gas analysis, studied 42 anthracycline

treated BC survivors (34 months post therapy cessation) and 11 age- and sex-matched controls.17

Breast cancer survivors had 21% lower VO2peak that was due to a lower peak Q secondary to a

lower peak SV with no significant difference found between groups for peak, mean arterial

pressure, systemic vascular resistance or estimated CaO2 – CvO2. Of note, resting SV was lower

while HR was higher resulting in a blunted HR reserve (peak minus rest value). A limitation of

impedance cardiography is that it is unable to evaluate the contribution of changes in left

ventricular (LV) systolic and diastolic volumes and function in limiting exercise SV.

A later study by Khouri et al., using echocardiography at rest and during post-exercise

recovery, equal to 83% of maximal HR, measured LV volumes and Q in 57 middle-aged

anthracycline treated BC survivors (51 years) and 20 age and sex-matched controls (57 years).16

Compared to controls, VO2peak was 20% lower in BC survivors with no significant difference

between groups for maximal exercise HR or SBP. Post-exercise Q was lower in BC survivors

secondary to a lower SV and EDV.16 A limitation of this study was that assessment of cardiac

function occurring in the recovery period.

17

Finally, Koelwyn et al.19, using 2D echocardiography during sub-maximal cycle exercise,

measured cardiac function in 30 anthracycline treated BC survivors and 30 age-, BMI- and

activity- matched women. In contrast to the above mentioned studies16, 17, VO2peak was not

significantly different between groups, and control subjects achieved greater peak systolic blood

pressure.19 Similar to prior studies16, 17, the BC group a had higher resting HR and both groups

achieved the same peak HR- suggesting reduced HR reserve in BC survivors. No differences

were found for LV EDV, ESV, SV or LVEF at rest, however, at 25, 50 and 75% of maximal

work rate, ESV was increased in BC survivors while LVEF reserve was blunted. These findings

were explained by impaired ventricular-arterial coupling in BC mediated by an increase in non-

invasively derived single-point end-systolic elastance (a surrogate for LV contractility).

Importantly, the estimated increase in end-systolic elastance may have been due to increased

myocardial stiffness due to fibrosis/extracellular matrix expansion, cardiomyocyte loss, or

impaired cardiomyocyte contractile function independent of LV contractility.

In an effort to better understand perturbations in end-systolic elastance, myocardial tissue

characteristics were characterized in a cross-sectional study of anthracycline treated cancer

survivors (n=37), non-anthracycline treated cancer survivors (n=17), pre-therapy cancer patients

(n=37), and cancer-free controls (n=236).20 Specifically, magnetic resonance imaging (MRI) was

used to non-invasively assess LV native T1 (an indicator of myocardial fibrosis) and T2

relaxation (a measure of myocardial edema). Previously, extracellular volume fraction (the ratio

of extracellular matrix to cardiomyocytes) was linked to cardiac tissue movement and diastolic

compliance.21 Consistent with findings of impaired end-systolic elastance from Koelwyn et al.19,

anthracycline treated cancer survivors had increased myocardial fibrosis (measured as T1 and

extracellular volume fraction with gadolinium) relative to cancer-free controls.20 However, pre-

18

treatment cancer patients also had elevated native T1, suggesting that even prior to therapy,

cancer patients may have increased LV chamber fibrosis and stiffness. T2 was within normal

ranges for both pre-treatment cancer patients and cancer survivors.

In summary, the role anthracycline induced cardiac impairment in reduced VO2peak is unclear.

Studies performed to date consistently demonstrate that BC survivors do not have a reduced peak

HR, however; resting HR is elevated and as a result, HR reserve is reduced.16, 17, 19 Some studies

have shown that peak Q is secondary to reduced SV16, 17, however studies in which LV volumes

were measured during exercise stress remain equivocal. Khouri et al.16 reported normal LVEF,

but were underpowered to detect subtle differences in LV volumes, while Koelwyn et al.19

reported a blunted LVEF reserve secondary to impaired ventricular-arterial coupling during sub-

maximal exercise. Further, the impaired ventricular-arterial coupling is consistent with studies

reporting increased myocardial extracellular volume fraction and fibrosis following

anthracycline therapy. However, there is evidence to suggest underlying differences in

myocardial tissue characteristics prior to therapy.20 There remains the possibility of reduced peak

Q prior to anthracycline therapy, suggesting underlying structural and/or functional cardiac

differences even in the absence of a cardiotoxic stimuli.

1.1 c) ii: Non-Cardiac Determinants of impaired VO2peak:

Non-cardiac peripheral abnormalities, may result in decreased oxygen delivery to and or

extraction by the active muscles during exercise. The volume of oxygen in a given volume of

arterial blood is described by the equation:

CaO2 = (SaO2%) x [Hb g/dl] x 1.34 ml O2/g Hb x blood volume

19

Where SaO2 is arterial oxygen saturation, Hb is hemoglobin, and 1.34 is a constant for the

oxygen carrying capacity of hemoglobin.

During peak exercise, there is no evidence to indicate reduced SaO2 in the absence of

overt pulmonary disease in BC. Although reduced pulmonary function is reported in BC and

linked to exercise capacity, reductions in pulmonary function do not contribute to reduced

arterial oxygen content.18

Anemia is a well-known consequence of anthracycline therapy22, 23, and can directly

lower the oxygen carrying capacity of blood. Reductions in hemoglobin are related to the change

in VO2 peak over the course of chemotherapy.24 In a 3-arm study comparing usual care (UC,

n=82), aerobic exercise training (AET, n= 78) and resistance exercise training (RET, n=82),

exercise did not attenuate declines in hemoglobin, but did preserve VO2 peak.24 In another study,

anemic cancer patients, including a subset of BC patients, were administered darbepoetin, a

synthetic stimulator of red blood cell production, with (n=26) or without (n=29) concomitant

AET. Peak oxygen uptake significantly improved in the AET group, and at study end,

hemoglobin trended to increase greater in the darbepoetin alone group (123.4±12.8 vs

120.6±16.3 g/l, p=0.067).25 Taken together, these data indicate a relationship between reduced

oxygen carrying capacity of blood and VO2peak, but do not support reduced oxygen carry capacity

of blood as a limiting determinant of exercise capacity. Further complicating the role of

anthracycline induced anemia are the findings by Kirshner et al. who reported that 31.3% of BC

patients had anemia prior to receiving chemotherapy22, a finding that suggests the possibility of

impaired VO2 peak in the absence of a cardiotoxic and anemic stimulus.

20

Given that CaO2 and SaO2 remain constant during exercise, a reduction in venous oxygen

saturation (SvO2%) drives increased oxygen extraction. SvO2% is a composite term dictated by

the Fick principle of diffusion:

Rate of Diffusion = k x A(P2 – P1)/D

Where k is a diffusion constant dependent upon solubility of the gas and temperature, A is the

surface area of gas exchange, (P2 – P1) is the diffusion gradient (partial pressure in mmHg) and D

is the diffusion distance.

Currently, no information is available directly linking perturbations involving factors

governing oxygen diffusion and VO2peak in anthracycline treated BC survivors. However, during

exercise, the predominant source of increased oxygen demand is in the active skeletal muscle.26

Accordingly, VO2 is determined by the mass of skeletal muscle that is active, the ability to

deliver oxygen to the muscle, and the ability of the muscle to utilize oxygen. In health, large

muscle mass exercise (>5-6 kg) is limited by oxygen delivery26, however, whether this is the

case in BC remains unknown.

In a seminal study by Demark-Wahnefried et al., BC patients receiving chemotherapy

(n=36, age: 41 years) were shown to gain weight, with a concomitant drop in skeletal muscle

mass from pre-to-post-therapy.27 This finding was linked to a ~25-75 kcal/day reduction in

physical activity energy expenditure, without a compensatory adjustment in caloric intake. Over

the course of one year (study end), BC patients treated with chemotherapy increased their fat

mass by 2.3 kg, and decreased their lean body mass by 0.5 kg, with lean body mass loss

predominantly localized to the thigh. While this study did not measure VO2peak, findings may be

inferred; absolute VO2 may decline as a result of decreased muscle mass, and relative VO2 would

21

decline due to a two-fold hit- increased fat mass and decreased active skeletal muscle mass.27

Indeed, therapy associated weight gain is a near ubiquitous finding, with a recent meta-analysis

demonstrating a pooled mean gain of 2.7 kg (95% CI: 2.0-3.3 kg) across 34 studies and 2620 BC

patients; increased adiposity is likely to be a contributing factor to reduced oxygen uptake in

BC.28

Reding et al. provided the first evidence of a link between anthracycline related changes

in body composition and declines in VOpeak.29 In a cross sectional study, 14 cancer survivors

(age: 54 years, 8 BC, >1 year post anthracycline therapy) and 14 sex- age- and BMI-matched

controls underwent magnetic resonance imaging (MRI) to characterize intermuscular fat (IMF)

and skeletal muscle (SM) in the paraspinal skeletal muscles at the level of the second lumbar

vertebra while cardiopulmonary exercise testing was performed to determine VO2 peak. Survivors

had 22% lower VO2peak, and trended to have greater paraspinal IMF (BC 13.9 ± 5.6 vs HC 11.7 ±

4.3 cm2, p=0.11) and IMF to SM ratio (BC 0.26 ± 0.10 vs HC 0.22 ± 0.10, p=0.13). In both

groups combined, VO2 peak was strongly and inversely related to the IMF:SM ratio (r -0.67,

p<0.01). Correlations persisted after adjustment for subcutaneous and visceral fat depots,

implicating a key role of IMF in reduced exercise capacity.29 The authors speculate that IMF

competes for blood flow and oxygen delivery to active muscle29, as has been proposed as a

mechanism of exercise intolerance in HF with preserved ejection fraction.30 Despite the strong

relationships reported by Reding et al., caution must be warranted as the paraspinal muscles play

a minimal role in VO2 compared to major locomotor muscles during peak whole body exercise.29

Mijwel et al. extend findings of skeletal muscle loss from pre-to post-BC chemotherapy

by measuring muscle fiber size and type, capillary to fiber ratio, and fiber oxidative capacity.31 In

a sample of 10 BC patients, muscle fiber cross-sectional area decreased, as well as the capillary

22

to fiber ratio and citrate synthase activity- the rate limiting enzyme in oxidative metabolism.

Markers of muscle atrophy were not increased, while markers of mitophagy were reduced, a

possible indicator of dysregulated quality control of mitochondria production. These changes

may contribute to a decrease in surface area available for oxygen exchange, reduced oxygen use

and therefore diffusion gradient, and potentially mixed effects on diffusion distance. Reduced

fiber cross sectional area would reduce diffusion distance from the cell membrane to

mitochondria, however, given fewer capillaries per muscle fiber, the diffusion distance from

capillary to mitochondria may be increased. In the same study, 13 BC patients underwent

exercise training which prevented a decrease in muscle fiber cross sectional area, capillary to

fiber ratio, and citrate synthase activity.31 How these findings translate to whole body VO2 is

unclear beyond skeletal muscle mass loss contributing to reduced VO2 peak as previously

discussed, however, reductions in muscle fiber oxidative capacity (citrate synthase activity)

could contribute to reduced whole body VO2 peak under the condition of adequate muscle oxygen

supply at peak exercise.

Muscle oxygen supply is determined by ability to deliver Q to active skeletal muscle.

Previously, vascular dysfunction has been hypothesized as a mechanism contributing to impaired

VO2 peak and elevated CV risk in BC, as vascular dysfunction is considered an incipient step in

the pathogenesis of CVD.32 An early study reported that over the course of anthracycline based

chemotherapy, vascular function measured as brachial artery flow mediated dilation (FMD) in

response to cuff ischemia did not decline (n=20, mean age=49).33 Results were later confirmed in

a larger sample (n=35), and in line with this finding, two, independent studies report no

difference in FMD between BC survivors (n=47, 30) and healthy controls (n=11, 30).17, 19, 34

Further, brachial artery function was not related to peak VO2.17 Taken together, evidence to date

23

suggests a minimal role of vascular (dys)function in limiting VO2 peak in anthracycline treated BC

survivors.

Vascular stiffening has also been proposed as a mechanism contributing to reduced

oxygen delivery (through impaired peak Q) and impaired exercise tolerance. In a case-control

study of cancer patients (BC n=19, lymphoma n=11, leukemia n=10) scheduled to receive

anthracycline based chemotherapy and control participants (n=13), aortic distensibility (relative

change in cross-sectional area of the artery normalized to pulse pressure) and pulse wave

velocity (PWV, pressure propagation speed) were measured as indicators of aortic stiffness.35

From pre to post therapy (4 months), PWV increased by 95%, with no corresponding change

over the same duration in the control group. Aortic distensibility consistently mirrored PWV

findings as distensibility dropped by more than 50% in cancer patients, and remained constant in

controls.35 In a follow-up study, Drafts et al. replicated PWV findings and provided time-course

information in 53 cancer patients (BC n=22, lymphoma n=17, leukemia n=13, myelodysplastic

syndrome n=1) undergoing anthracycline based chemotherapy.36 Pulse wave velocity was

measured prior to the start of therapy, and at 1, 3 and 6 months; PWV increased steadily from

baseline (6.7±0.5) to 6 months (10.1±1 m/s). In contrast, a recent study of 35 BC patients found

that over the course of anthracycline based therapy carotid-femoral, carotid-femoral PWV

decreased (16.7±11.8 to 14.9±8.4 m/s, n.s.), while aortic compliance (assessed by ultrasound) did

not change (0.061±0.04 to 0.046±0.03 mm/mmHg, n.s.).34 Taken together, these findings

indicate potential structural vascular involvement in the determination of VO2 peak. Despite mixed

longitudinal findings, it should be noted that prior to anthracycline based chemotherapy, studies

consistently report aberrant vascular stiffness, suggesting underlying structural vascular changes

24

independent of potentially vascular-toxic anthracycline therapy.34-36 Whether increased vascular

stiffness limits blood flow to working muscle and potentially impairs VO2 peak was not addressed.

To date, only one study has investigated the ability to deliver blood to working muscle.37

Small muscle mass exercise can be used as a paradigm to isolate ability to deliver blood to active

muscle, as metabolic demands of small muscle mass exercise do not approach the limits of peak

Q.38 In a mixed cohort of cancer survivors (n=11, BC n=10) treated with adjuvant therapy

(anthracycline based n=4), ultrasound was used to assess forearm blood flow in response

rhythmic handgrip exercise at 20% of maximal voluntary contraction.37 Cancer survivors

achieved significantly lower forearm blood flow and mean arterial pressure during exercise, with

no difference in forearm vascular conductance between groups. Despite using a paradigm

designed to remove the heart as a limiting component of exercising blood flow, the authors

attribute the finding of reduced forearm blood flow to an impaired blood pressure response,

secondary to reduced LV ejection time (derived from the arterial pressure waveform, a measure

of ventricular systolic performance).37 The blunted forearm blood flow response could not be

attributed to different relative or absolute workloads, raising the possibility that given the same

absolute workload and oxygen demand for the given workload, lower forearm blood flow must

be compensated for by increased oxygen extraction. In the context of whole body VO2, the same

effect would imply preserved VO2peak despite any cardiac or vascular perturbations associated

with BC.

1.1 d) Statement of the problem:

The pathophysiology of exercise intolerance in anthracycline treated BC is not well

understood. Anthracyclines have clearly defined cardiotoxicity, however, only a small portion of

anthracycline treated BC survivors have evidence of subclinical cardiac dysfunction at rest, and

25

the sparse literature available leaves the possibility of underlying cardiac impairment open.11, 16,

17 Further, the paucity of studies that have examined isolated (cardiac, vascular and skeletal

muscle function) components of the oxygen cascade have not examined components during

maximal exercise.

The following studies aim to 1) develop a clinically viable MRI exercise stress imaging

technique to assess cardiac function during exercise (Chapter 2); 2) examine exercising cardiac

function in BC patients prior to anthracycline therapy as a determinant of VO2 peak (Chapter 3); 3)

examine peripheral hemodynamic responses during submaximal single leg knee extension

(SLKE) exercise in BC survivors and controls (Chapter 4); and 4) examine muscle composition,

lower leg blood flow, oxygen utilization and bioenergetics as potential factors contributing to

exercise intolerance in BC survivors (Chapters 5). Taken together, these studies aim to elucidate

pathophysiological mechanisms underpinning reduced cardiorespiratory fitness in anthracycline

treated BC.

26

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27

17. Jones LW, Haykowsky M, Pituskin EN, Jendzjowsky NG, Tomczak CR, Haennel RG and

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19. Koelwyn GJ, Lewis NC, Ellard SL, Jones LW, Gelinas JC, Rolf JD, Melzer B, Thomas SM,

Douglas PS, Khouri MG and Eves ND. Ventricular-Arterial Coupling in Breast Cancer Patients After

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20. Jordan JH, Vasu S, Morgan TM, D'Agostino RB, Jr., Melendez GC, Hamilton CA, Arai AE, Liu

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Hemoglobin and Aerobic Fitness Changes with Supervised Exercise Training in Breast Cancer Patients

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Mackey JR. Effects of aerobic exercise training in anemic cancer patients receiving darbepoetin alfa: a

randomized controlled trial. Oncologist. 2008;13:1012-20.

26. Poole DC and Richardson RS. Determinants of oxygen uptake. Implications for exercise testing.

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27. Demark-Wahnefried W, Peterson BL, Winer EP, Marks L, Aziz N, Marcom PK, Blackwell K and

Rimer BK. Changes in weight, body composition, and factors influencing energy balance among

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28. van den Berg MM, Winkels RM, de Kruif JT, van Laarhoven HW, Visser M, de Vries JH, de

Vries YC and Kampman E. Weight change during chemotherapy in breast cancer patients: a meta-

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29. Reding KW, Brubaker P, D’Agostino R, Kitzman DW, Nicklas B, Langford D, Grodesky M and

Hundley WG. Increased skeletal intermuscular fat is associated with reduced exercise capacity in cancer

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30. Haykowsky MJ, Kouba EJ, Brubaker PH, Nicklas BJ, Eggebeen J and Kitzman DW. Skeletal

muscle composition and its relation to exercise intolerance in older patients with heart failure and

preserved ejection fraction. Am J Cardiol. 2014;113:1211-6.

31. Mijwel S, Cardinale DA, Norrbom J, Chapman M, Ivarsson N, Wengstrom Y, Sundberg CJ and

Rundqvist H. Exercise training during chemotherapy preserves skeletal muscle fiber area, capillarization,

and mitochondrial content in patients with breast cancer. FASEB journal : official publication of the

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32. Widmer RJ and Lerman A. Endothelial dysfunction and cardiovascular disease. Global

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28

33. Jones LW, Fels DR, West M, Allen JD, Broadwater G, Barry WT, Wilke LG, Masko E, Douglas

PS, Dash RC, Povsic TJ, Peppercorn J, Marcom PK, Blackwell KL, Kimmick G, Turkington TG and

Dewhirst MW. Modulation of Circulating Angiogenic Factors and Tumor Biology by Aerobic Training in

Breast Cancer Patients Receiving Neoadjuvant Chemotherapy. CANCER PREVENTION RESEARCH.

2013;6:925-937.

34. Mizia-Stec K, Goscinska A, Mizia M, Haberka M, Chmiel A, Poborski W and Gasior Z.

Anthracycline chemotherapy impairs the structure and diastolic function of the left ventricle and induces

negative arterial remodelling. Kardiol Pol. 2013;71:681-90.

35. Chaosuwannakit N, D'Agostino R, Hamilton CA, Lane KS, Ntim WO, Lawrence J, Melin SA,

Ellis LR, Torti FM, Little WC and Hundley WG. Aortic Stiffness Increases Upon Receipt of

Anthracycline Chemotherapy. JOURNAL OF CLINICAL ONCOLOGY. 2010;28:166-172.

36. Drafts BC, Twomley KM, D'Agostino R, Jr., Lawrence J, Avis N, Ellis LR, Thohan V, Jordan J,

Melin SA, Torti FM, Little WC, Hamilton CA and Hundley WG. Low to moderate dose anthracycline-

based chemotherapy is associated with early noninvasive imaging evidence of subclinical cardiovascular

disease. JACC Cardiovasc Imaging. 2013;6:877-85.

37. Didier KD. Altered Blood Flow Response to Small Muscle Mass Exercise in Cancer Survivors

Treated With Adjuvant Therapy. 2017;6.

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exercise: a hierarchy of competing physiological needs. Physiol Rev. 2015;95:549-601.

2.1 Exercise Cardiac Magnetic Resonance Imaging: A feasibility study and Meta-

Analysis.

Rhys I. Beaudry1, T. Jake Samuel1, Jing Wang2, Wesley J. Tucker1, Mark J. Haykowsky2,

Michael D. Nelson1, Exercise Cardiac Magnetic Resonance Imaging: A feasibility study and

Meta-Analysis. Am J Physiol Regul Integr Comp Physiol. 2018 315:4, R638-R645;

https://doi.org/10.1152/ajpregu.00158.2018

1Department of Kinesiology, University of Texas at Arlington, 500 W. Nedderman Drive,

Arlington, TX 76013, USA

2College of Nursing and Health Innovation, University of Texas at Arlington, 411 S. Nedderman

Drive, Arlington, TX 76010, USA

Used with permission of the publisher, 2019.

30

2.1 a) Abstract

Cardiac stress testing improves detection and risk assessment of heart disease. Magnetic

resonance imaging (MRI) is the clinical gold-standard for assessing cardiac morphology and

function at rest; however, exercise MRI has not been widely adapted for cardiac assessment due

to imaging and device limitations. Commercially available MR ergometers, together with

improved imaging sequences, have overcome many previous limitations, making cardiac stress

MRI more feasible. Here, we aimed to demonstrate clinical feasibility, and establish the

normative, healthy response to supine exercise MRI. Eight young, healthy subjects, underwent

rest and exercise cinematic imaging to measure left ventricular volumes and ejection fraction. To

establish the normative, healthy response to exercise MRI we performed a comprehensive

literature review and meta-analysis of existing exercise cardiac MRI studies. Results were pooled

using a random effects model to define the left ventricular ejection fraction, end-diastolic, end-

systolic, and stroke volume responses. Our proof-of-concept data showed a marked increase in

cardiac index with exercise, secondary to an increase in both heart rate and stroke volume. The

change in stroke volume was driven by a reduction in end-systolic volume, with no change in

end-diastolic volume. These findings were entirely consistent with 17 previous exercise MRI

studies (226 individual records), despite differences in imaging approach, ergometer, or exercise

type. Taken together, the data herein demonstrate that exercise cardiac MRI is clinically feasible,

using commercially available exercise equipment and vendor-provided product sequences, and

establish the normative, healthy response to exercise MRI.

31

2.1 b) New & Noteworthy:

Cardiac stress testing improves detection and risk assessment of heart disease; however,

exercise MRI has not been widely adapted for cardiac assessment due to imaging and device

limitations. Here, we demonstrate clinical feasibility of cardiac stress MRI using a commercially

available MRI compatible ergometer and vendor provided product sequences. Moreover, by

performing a comprehensive literature review and meta-analysis of existing exercise cardiac

MRI studies, we establish the normative, healthy response to supine exercise MRI.

32

2.1 c) Introduction

Exercise stress testing provides important prognostic information for assessing

cardiovascular risk and detection of heart disease 39-45. Combining exercise stress testing with

cardiac imaging, such as echocardiography, nuclear imaging or positron emission tomography,

significantly improves diagnostic sensitivity and specificity 46-50. Accordingly, exercise stress

imaging is commonly performed world-wide 51-55.

Cardiac magnetic resonance imaging (cMRI) is the clinical gold-standard for assessing

cardiac morphology and global systolic function 56-58. Despite its excellent spatial resolution, and

unique potential for additional structural and functional quantification (e.g. fibrosis assessment

and/or myocardial energetics), cMRI is not currently utilized for cardiac exercise stress imaging.

While many factors may contribute to the underutilization of stress cMRI, the general lack of

MR compatible exercise equipment and inadequate imaging sequences likely play a major role

59.

To overcome these limitations, several investigators have advocated exercising outside of

the MRI followed by a brief transition to the scan table and subsequent imaging 60-68. Under this

paradigm, with much familiarization and the use of innovative body molds (to ensure anatomical

image registration), image acquisition can be performed within 60 seconds of peak exercise 63.

Marked hemodynamic recovery during the transition from exercise to imaging 60-63, 67, 68, and the

technical training and familiarization required of both the patient and imaging staff to achieve

rapid and accurate body placement, has prevented wide spread adoption of this approach.

Accordingly, several investigators have focused on MRI compatible cycle ergometers, which

allow patients to exercise inside the bore of the magnet 69-78. While several studies have

33

evaluated cardiac output dynamics using velocity encoding approaches 70, 79-85, only a few studies

have attempted to evaluate ventricular morphology and function 69, 73, 75, 77, 78, 86-94; the vast

majority of which have utilized a real-time, ungated free-breathing pulse sequence 73, 87-90, 94.

While this latter approach increases overall feasibility by eliminating ECG-gating artifacts,

patient breath-holds, and minimizes the impact of chest movement, post-processing is incredibly

complex and time consuming, negating the possibility of “real-time” image reconstruction during

the cardiac stress test (and thus real-time physician feedback). Such limitations have also

prevented the wide-spread adoption of this approach.

With this background, and the goal of making exercise cMRI clinically translatable, the

primary aim of this study was to test the feasibility of using a commercially available MR

compatible ergometer, combined with vendor provided MR imaging sequences, to assess cardiac

morphology and function. To assess the impact of movement on image quality, we compared

images acquired during exercise, with images acquired immediately (0-4 seconds) post-exercise.

To assess the impact of a prolonged pause between exercise and imaging—similar to that

encountered when exercise is performed outside of the bore of the MRI— we compared subject

hemodynamics, cardiac morphology and cardiac function between exercise and following a 60-

second pause. A secondary aim of this study was to perform a comprehensive literature review

and meta-analysis of published studies reporting cardiac volumes during exercise cMRI to

establish the normative response.

2.1 d) Methods

34

2.1 d) i: Participants

Eight recreationally active, young, healthy subjects were recruited from the Dallas-Fort

Worth community. Participants were eligible for inclusion if they were 18–35 years old,

physically active, and had no history of cardiovascular, metabolic or neurological disease.

Exclusion criteria included: contraindications to MRI and physical limitations precluding

exercise.

All procedures were performed in accordance with the principles of the Helsinki

declaration; all participants provided written informed consent and the study was approved by

the University of Texas at Arlington and University of Texas Southwestern Medical Center

Institutional Review Boards.

2.1 d) ii: Protocol To address our primary aim, each subject completed three visits. The first visit was used

for screening, familiarization with the MRI compatible ergometer (Ergospect Cardio-Stepper,

Ergospect, Austria), and determination of target workload and heart rate for exercise cMRI. The

second visit was used to characterize the metabolic cost and hemodynamic differences between

supine exercise with our MR compatible ergometer and upright exercise with a traditional cycle

ergometer (Lode Corival, Lode, Netherlands). The third visit consisted of a resting and exercise

stress cMRI.

Supine Cardiopulmonary Exercise Test: Subjects underwent a supine, incremental

maximal exercise test, starting at 15 watts at a cadence of 50 revolutions per minute (RPM) and

progressing by 15 watts every 2 minutes until volitional exhaustion or significant and continued

loss of cadence (>10 RPM despite verbal encouragement to continue). Heart rate (Polar H1,

35

Polar, USA), expired gas analysis (TrueOne 2400, Parvomedics, USA) and work-rate (Ergospect,

Cardio-Stepper, Ergospect Austria) were recorded continuously.

Upright Cardiopulmonary Exercise Test: Subjects underwent an incremental ramped

maximal exercise test, starting at 50 watts at a self-selected cadence (50-90 RPM) and

progressing by 20 watts every 2 minutes until volitional exhaustion. Heart rate (Polar H1, Polar,

USA), expired gas analysis (TrueOne 2400, Parvomedics, USA) and work-rate (Lode Corival,

Lode, Netherlands) were recorded continuously.

Cardiac MRI: Imaging was conducted on a Phillips Achieva 3T scanner. Following

resting imaging, subjects exercised at the workload, determined from study visit 1, required to

achieve 4 metabolic equivalents (METs, 4 METs ≈ 14 ml/kg/min); the minimal threshold for

independent living 95. High resolution long-axis (4-chamber and 2 chamber) and short axis (mid-

ventricular, papillary muscle level) cine images were acquired using balanced fast field echo at

rest, during exercise, immediately (0-4 seconds) post-exercise, and 60-seconds post-exercise (to

mimic patient transfer from an external exercise device). Typical image parameters include: TR

= 3.4 msec; TE = 1.7 msec; flip angle = 45º; acquisition matrix = 195 x 195; field of view = 295

x 295 mm; 8-mm slice thickness; 20-30 phases/cardiac cycle; with one slice acquired per breath

hold. Breath-hold time ranged between 7-9 seconds at rest and 3-5 seconds during exercise.

Cardiac MRI data were analyzed using commercially available software (CVI42, Circle

Cardiovascular Imaging Inc., Canada). LV volumes were calculated by Simpson’s Biplane

method using standard 4 and 2-chamber slices, and cross-sectional area was measured in the

short axis plane. All volumes were indexed to body surface area; calculated by the Dubois-

Dubois formula, to account for body size variation 96.

36

2.1 d) iii: Comprehensive Literature Review

To accomplish our secondary aim, we performed a comprehensive literature review,

searching articles on PubMed from 1985 to March 2018, using the search terms “exercise AND

cardiac MRI”, hand searched references, and contacted experts in the field. A priori, we included

studies reporting left-ventricular cardiac volumes for healthy controls aged 16-35, to best match

our participant inclusion criteria. We extracted mean resting and exercise end-diastolic, end-

systolic and stroke volumes, heart rates, and ejection fractions from study text, tables and figures

(WebPlotDigitizer, V 4.1, Rohatgi, USA). If data were not readily extractable, authors were

contacted directly and asked to provide the missing or unattainable data.

2.1 d) iv: Statistical Analysis

Aim 1 – Feasibility Study: Dependent variables were confirmed for normality and

homoscedasticity using the Shapiro-Wilk test. Changes in cardiac volumes and global left

ventricular function from rest to exercise/post-exercise were assessed using a one-way repeated

measures ANOVA with Sidak post hoc testing. Hemodynamic and cardiopulmonary data were

compared between the supine and upright incremental exercise tests using a paired sample t-test.

Statistical analyses were performed using SPSS (version 24 IBM SPSS Statistics, Armonk,

USA). All data are expressed as mean (standard deviation) unless otherwise stated, and statistical

significance was considered when P ≤ 0.05.

Aim 2 – Comprehensive Literature Review: Previously published results from the

literature review were pooled and analyzed by a random-effect analysis to create a single, more

precise estimate of the effect size. The rest/exercise correlation was assumed to be moderate

(50%) 97. Analysis and graphical presentation were performed in R 3.4.2 using the “metaphor”

37

package (R Core Team (2016), R Foundation for Statistical Computing, Vienna, Austria). Effect

sizes were calculated using both Standardized Mean Differences (SMD) and Weighted Mean

Differences (WMD) between rest and exercise conditions. A SMD of 0.25 was considered as a

small effect size, 0.5 as a medium effect size, and 0.8 and higher as a large effect size.

Heterogeneity of studies was explored using the Cochrane’s Q test of heterogeneity (P < 0.05

was considered statistically significant). Inconsistency in the results of the studies was assessed

by I2 which described the percentage of total variation across studies that was due to

heterogeneity. When I2 was > 50%, there was more than moderate inconsistency 98, 99.

2.1 e) Results

2.1 e) i: Aim 1 – Feasibility Study

Eight healthy men and women completed the feasibility study (M/F, 3/5; age, 25 + 3

years; BMI, 22.3 + 2.7 kg/m2; BSA, 1.62 + 0.14 m2).

Supine vs. Upright Exercise: The metabolic and hemodynamic data for both the supine

and upright cardiopulmonary stress tests are displayed in Table 1. Peak oxygen consumption

was markedly lower during supine exercise compared to upright cycling, reflective of the smaller

muscle mass being utilized with the MR compatible ergometer. Similarly, peak heart rate, peak

respiratory exchange ratio, and peak ventilation were also markedly lower. None of the subjects

met the criteria for true VO2max during supine exercise, defined as achieving: 1) age predicted

maximal heart rate; 2) respiratory exchange ratio > 1.10; or 3) a plateau in oxygen consumption.

In contrast, all of the subjects during the upright cycling ramp test met at least two of these

criteria. The primary reason for stopping the supine exercise test was an inability to overcome

38

the change in resistance; none of the subjects reported maximal perceived exertion or

breathlessness as a reason for stopping.

cMRI Feasibility: Cardiac imaging was possible during exercise in all of the subjects.

None of the subjects reported any discomfort or experienced any adverse events. As expected,

image quality was improved post-exercise (0-4 second, 60 seconds), when upper body and chest

coil movement were eliminated (Figure 1).

Both heart rate and stroke volume increased with exercise, resulting in a 2-fold increase

in cardiac index (CI) (Figure 1). The increase in stroke volume was mediated entirely by a

reduction in end-systolic volume (ESV), as end-diastolic volume (EDV) was unchanged with

exercise. Accordingly, ejection fraction was also significantly increased with exercise.

As compared to imaging during exercise, imaging immediately post-exercise had no

significant impact on chamber volumes, ejection fraction or cardiac index. In contrast, imaging

60-seconds post-exercise resulted in a significant increase in end-systolic volume, and a

significant decrease in heart rate, ejection fraction, and cardiac index (Figure 1).

2.1 e) ii: Aim 2 - Literature Review and Data Synthesis

Our search produced 1787 articles; 17 studies reported rest and exercise cardiac volumes.

Of the 17 studies, data were extracted from 14 studies (Table 2). One study 72 reported median

and interquartile range which could not be used in our synthesis. Two studies 77, 100 were

confirmed to have used previously reported data sets and were therefore excluded. . When

multiple exercise intensities were reported, the exercise intensity closest to that chosen in our

feasibility study was reported.

39

Characteristics of the studies included in our comprehensive data synthesis are displayed

in Table 2. The SMD model was used to account for variance in study participants (sex, age,

BSA, exercise capacity) and volumetric reporting (standard versus normalized volumes). By

combining these datasets, we were able to evaluate the normal physiological response to supine

exercise in 226 healthy subjects. The pooled analysis support our proof-of-concept data (reported

above), showing a marked and significant increase in ejection fraction during exercise compared

to rest (Figure 2 and Table 3). Moreover, we observed no change in left ventricular end-

diastolic volume, with changes in stroke volume being entirely driven by reductions in end-

systolic volume (Figure 2 and Table 3). Similarly, the magnitude of change in cardiac index

was therefore predominantly driven by changes in heart rate (Table 2). The Q test revealed

significant variation in the exercise response to ejection fraction (P = 0.004), end-systolic

volume (P = 0.007), and stroke volume (P = 0.005), although less than moderate (I2 = 41%, 49%

and 27%). There was no heterogeneity in end-diastolic volume response (Table 3A).

Visual inspection of Figure 2 prompted post-hoc analysis to determine if the Lafountain

study (2016, treadmill exercise) was a statistical outlier. Based on our model, approximately 5%

of the externally standardized residuals would be expected to exceed the bounds ±1.96.

Externally standardized residuals were calculated for end-systolic volume, stroke volume and

ejection fraction as -3.85, 1.92 and 12.24 respectively, indicating the study as a potential outlier.

In a sensitivity analysis, removal of Lafountain 2016 from the meta-analysis increased Q test p-

values (0.08, 0.05 and 0.26) and reduced I2 values (38%, 42%, and 26%) for ESV, SV and EF,

demonstrating a significant reduction in heterogeneity.

2.1 f) Discussion

40

The major novel findings of this study are three-fold: First, we demonstrate feasibility of

exercise stress cMR using a commercially available leg ergometer, and vendor-provided product

sequences at 3T. Second, we demonstrate hemodynamic similarities between imaging during and

immediately post-exercise, but marked hemodynamic decline when imaging is delayed for at

least 60-seconds post exercise. Finally, by combining our results with previously published data,

we were able to define the normal cardiac response to exercise cMRI.

That cardiac imaging was feasible using a commercially available leg ergometer, and

vendor-provided product sequences, provides great promise for easy and simple adoption of

exercise cMRI in clinical settings. While we observed a marked improvement in image quality

by imaging immediately post-exercise, we observed no major differences in cardiac volumes or

global left ventricular function between these two conditions. Because inclusion of brief pauses

will prolong the exercise stress imaging protocol, we believe imaging during exercise will prove

to be the protocol of choice for most clinical sites. Of course, if exercise intensity is increased

beyond the submaximal level performed herein, imaging during brief periods of exercise

cessation may need to be adopted to overcome the anticipated upper body and chest coil

movement. Indeed, as movement artifact increases with higher exercise intensities, spatial

resolution may degrade despite consistent imaging parameters. Caution is indeed warranted

when interpreting data acquired past 4-seconds post-exercise however, as our results suggest,

prolonged periods of rest following exercise are associated with sharp hemodynamic recovery.

While it may be argued that the rapid hemodynamic recovery observed in this study is reflective

of the younger, healthy population studied herein, clinical patients exercised outside of scanner

bore and transferred for imaging share a comparable decline in heart rate 63, 67.

41

Our feasibility data, together with our meta-analysis, demonstrate that the increase in

cardiac output during submaximal supine exercise is driven predominantly by an increase in

heart rate, with minimal contribution from stroke volume. The data overwhelmingly suggest that

healthy subjects have no end-diastolic volume reserve during supine exercise, despite the

expected increase in respiratory and muscle pumps. That this observation is consistent across

studies which did not perform breath holds 68, 73, 87-90 argues against intrathoracic pressure (i.e.

Valsalva maneuver) mediated reductions in preload. These data must therefore reflect the fact

that supine positioning, combined with leg elevation (feet in the ergometer stirrups), produces

near maximal end-diastolic volumes.

That we only observed a small reduction in end-systolic volume with exercise was

surprising, though consistent with other cMRI studies in healthy populations 68, 69, 73, 75, 78, 86-94.

Admittedly, this investigation was focused on submaximal exercise, and thus we cannot extend

these end-systolic volume findings to higher exercise intensities. Indeed, studies which included

higher workloads have reported exercise intensity dependent reductions in end-systolic volume

72, 73, 87-90. This inter-study difference likely contributed to the heterogeneity of findings in our

meta-analysis for end-systolic volume, ejection fraction, and stroke volume. Regardless of

exercise intensity however, the absolute change in end-systolic volume remains fairly low. This

may reflect the fact that supine exercise with MR compatible ergometers may not recruit

sufficient muscle mass to challenge cardiac output enough to demand greater end-systolic

volume changes. Compared to conventional upright cycling, our data clearly show blunted

metabolic demand; also observed in a prior investigation using a different MR compatible

ergometer 72.

42

This proof-of-concept feasibility study is not without limitation. First, we limited our data

collection to young healthy participants. Future studies are needed to extend these data to clinical

populations. Second, spatial resolution was purposely sacrificed to shorten breath-hold time. As

a result, the cine images were not sufficient for tissue deformation (i.e. strain) analysis. Inclusion

of quantifiable regional wall motion measurements will be an important future addition,

especially in clinical populations.

2.1 g) Conclusion

Taken together, the data herein establish the proof-of-concept that exercise cMRI can be

easily adapted to a clinical setting, providing gold-standard non-invasive stress images during

exercise. Using commercially available MRI compatible exercise equipment and vendor-

provided product sequences, we were able to successfully characterize the cardiac response to

submaximal exercise. By combining our feasibility data with previously published results, we

also establish the normative cardiac stress response in over 200 healthy subjects. With this

background in place, future studies are needed to adapt this approach in clinical populations.

43

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48

2.1 i) Figure Legends:

2.1 i) i: Figure 1: A) Representative high resolution cMR images at rest (black circle), during exercise (yellow

square), immediately post-exercise (0-4 seconds, green triangle) and 60 seconds post-exercise

(red diamond). Left ventricular volumes were indexed to body surface area for determining LV

end-diastolic (B), end-systolic (C) and stroke volume (D) and multiplied by heart rate (E) to

generate cardiac index (I). LV cavity areas were measured from a mid-short-axis image

(papillary muscle level), and indexed to body surface area, for determination of end-diastolic (F)

and end-systolic (G) cavity areas. Ejection fraction (H) was calculated from the biplane volumes.

Data presented as mean and standard error: † indicates a statistically significant (p<0.05)

difference from rest, and ‡ from exercise.

2.1 i) ii: Figure 2: Pooled standardized mean difference (exercise-rest, [95% confidence interval] for left ventricular

end-diastolic (A), end-systolic (B), and stroke volume (C), and ejection fraction (D) from the

available exercise cMR studies. 1Values taken from graphical representations at moderate, and

2peak intensity exercise. 3Adolescent populations, with 4values taken from graphical

representations.

49

2.1 j) Tables

2.1 j) i: Table 1: Metabolic and hemodynamic comparison between supine and

upright exercise. Table 1: Metabolic and hemodynamic comparison between supine and upright exercise

Parameter Supine Upright Percent

of upright

P value

VO2peak (ml/kg/min) 26 (3) 39 (5) 67 (10) 0.0007

VO2peak (l/min) 1.24 (0.35) 2.31 (0.53) 67 (10) 0.0004

Ventilationpeak, (l/min) 57 (15) 106 (24) 56 (13) 0.0003

RERpeak 1.07 (0.06) 1.23 (0.09) 87 (6) 0.0007

Heart rate (bpm) 153 (19) 188 (9) 82 (9) 0.0011

VO2peak, peak oxygen consumption; RER, respiratory exchange ratio.

50

2.1 j) ii: Table 2: Characteristics of exercise cMRI studies reporting LV

volumes.

Table 2: Characteristics of exercise cMRI studies reporting LV Volumes

HR, heart rate; NR, not reported; SAX, short axis; LAX, long axis. *Sub-maximal data used

in meta-analysis, see Figure 2 legend.

Study n

(m/f)

Age Resting

HR

Image

HR

Exercise

Intensity

Field

Strength

Exercise Type Image Type

Present Study 3/5 25 (3) 67 (8) 115 (9) Sub-Max 3.0T Supine, Ergospect

CardioStepper

Gated, breath-hold,

SAX, Bi-plane

LAX

Claessen

2014(a)89

14/0 36 (6) 66 (7) 143 (7) Max* 1.5T

Supine, Lode MRI

cycle

Real-time free-

breathe SAX/LAX

stacks Claessen

2014(b)88

8/1 35

(12)

NR NR Max*

Claessen

201590

14/0 36

(15)

66 (7) 149 (11) Max

Gusso 201269 4/4 25 (4) 67 (15) 110 (3) Sub-Max 1.5T

Supine, Custom

MRI cycle

Gated breath-hold

SAX stack, 3 LAX Gusso 201291

(2017)100

10/12 17 (1) 71 (9) 109 (5) Sub-Max

La Gerche

201273

10/0 35 (9) 59 (14) 164 (10) Max* 1.5T Supine, Lode MRI

cycle

Real-time free-

breathe SAX/LAX

stacks

LaFountain

201668

5/5 26 (5) 62 (8) 166 (20) Max* 1.5T Upright, Custom

MRI treadmill

Ungated, free-

breathe SAX stack

and biplane LAX

slices

Lurz 200994 5/7 33

(29-

42)

74 (3) 154 (13) Max* 1.5T Supine, Lode MRI

cycle, flutter

kicking motion

Real-time free-

breathe SAX

stacks

Oosterhof

200578

8/6 27 (4) 66 (8) 121 (8) Sub-Max 1.5T Supine, Custom

MRI cycle

Turbo field echo

planar imaging

SAX stack

Pinto 201492 10/9 17 (2) 70 (10) 109 (4) Sub-Max 1.5T Supine, Custom

MRI cycle

Gated breath-hold

SAX stack, 3 LAX

Roest 200186

(2002)77

8/8 18 (2) 71 (10) 121 (14) Sub-Max 1.5T Supine, Custom

MRI ergometer

Gated breath-hold

SAX stack

Roest 200493 7/7 25 (5) 67 (8) 122 (8) Sub-Max

Schnell

2017(a)87

15/0 35 (8) 59 (12) 155 (18) Max 1.5T Supine, Lode MRI

cycle

Real-time free-

breathe SAX/LAX

stacks Schnell

2017(b)87

12/3 35

(14)

66 (7) 150 (12) Max

Steding-

Ehrenborg

201375

20/6 30 (8) 60 (11) 94 (13) Sub-Max 1.5T Supine, Custom

MRI ergometer

Gated breath-hold

SAX stack and

LAX slice

51

2.1 j) iii: Table 3. Effect size, significance, and heterogeneity for pooled

analyses. Table 3: Meta-Analysis effect size, significance and heterogeneity

A. Measure n

(comparisons)

SMD

[95% CI]

Significance

(H0:SMD = 0)

Q

p-value

I2

(%)

EDV 16 -0.14 [-0.31, 0.04] 0.13 1.00 0

ESV 16 -1.18 [-1.42, -0.95] <0.0001 0.005 41

SV 16 0.87 [0.64, 1.09] <0.0001 0.006 50

EF 15 1.75 [1.48, 2.03] <0.0001 0.006 26

B. WMD (H0:WMD = 0)

EDV (ml) 11 -0.33 [-0.61, -0.06] 0.027 0.18 28

ESV (ml) 11 -3.51 [-4.11, -2.91] <0.0001 <0.0001 75

SV (ml) 11 3.86 [3.07, 4.65] <0.0001 <0.0001 91

EF (%) 15 6.12 [5.26, 6.98] <0.0001 0.005 95

SMD, standardized mean difference; WMD, weighted mean difference; 95% CI, 95% confidence

intervals; EDV, end-diastolic volume; ESV, end-systolic volume; SV, stroke volume; EF,

ejection fraction. Note: SMD calculated by pooling EDV (ESV, SV) for studies reporting

measures in ml, ml/m2 (BSA normalized) and ml/kg (normalized to fat free mass). WMD

calculated by pooling studies reporting EDV (ESV, SV) in ml.

52

2.1 k) Figures

Figure 1: cMRI Images and Results

53

Figure 2: Meta-Analysis Results

54

3.1 Determinants of Exercise Intolerance in Breast Cancer Patients Prior to

Anthracycline Chemotherapy

Rhys I. Beaudrya, Erin J. Howdenb, Steve Foulkesb,c, Ashley Bigaranb,d, Mark J. Haykowskya,b*,

Andre La Gercheb,e* Determinants of exercise intolerance in breast cancer patients prior to

anthracycline chemotherapy. Physiol Rep. 2019 7(1):e13971. doi: 10.14814/phy2.13971

*contributed equally as senior authors.

aIntegrated Cardiovascular Exercise Physiology and Rehabilitation Laboratory, College of

Nursing and Health Innovation, University of Texas at Arlington, Arlington, Texas, USA

bSports Cardiology Lab, Baker Heart and Diabetes Institute, Melbourne, Vic, Australia;

cSchool of Exercise & Nutrition Sciences, Deakin University Faculty of Health, Burwood, Vic,

Australia;

dExercise and Nutrition Research Program, Mary McKillop Institute for Health Research,

Australian Catholic University, Melbourne VIC, Australia.

eDepartment of Cardiology, St Vincent’s Hospital Melbourne, Fitzroy, Vic, Australia

Used with permission of the publisher, 2019.

55

3.1 a) Abstract

Background: Women with early stage breast cancer have reduced peak exercise oxygen uptake

(peak VO2). The purpose of this study was to evaluate peak VO2 and right (RV) and left (LV)

ventricular function prior to adjuvant chemotherapy.

Methods: Twenty-nine early stage breast cancer patients (mean age: 48 years) and 10 age-

matched healthy women were studied. Participants performed an upright cycle exercise test with

expired gas analysis to measure peak VO2. RV and LV volumes and function were measured at

rest, sub-maximal and peak supine cycle exercise using cardiac magnetic resonance imaging.

Results: Peak VO2 was significantly lower in breast cancer patients versus controls (1.70.4 vs.

2.30.5 l/min, p=0.0013; 256 vs. 356 ml/kg/min, p=0.00009). No significant difference was

found between groups for peak upright exercise heart rate (17413 vs. 16916 bpm, p=0.39).

Rest, sub-maximal and peak exercise RV and LV end-diastolic and end-systolic volume index,

stroke index, and cardiac index were significantly lower in breast cancer patients versus controls

(p<0.05 for all). No significant difference was found between groups for rest and exercise RV

and LV ejection fraction.

Conclusion: Despite preserved RV and LV ejection fraction, the decreased peak VO2 in early

stage breast cancer patients prior to adjuvant chemotherapy is due in part to decreased peak

cardiac index secondary to reductions in RV and LV end-diastolic volumes.

56

3.1 b) Introduction

Breast cancer is the most frequently diagnosed malignancy among women and the second

leading cause of cancer mortality in the United States.101 During the past three decades, the

breast cancer mortality rate has decreased by nearly 40% as a result of advances in prevention,

early detection and treatment.2 Despite improved survival, breast cancer survivors are at

increased at risk of developing cardiovascular disease that is due, in part, to an unhealthy

lifestyle associated with sedentary deconditioning.102, 103 103-106

Breast cancer patients have severely reduced exercise tolerance, measured objectively as

decreased peak oxygen uptake (peak VO2), that is associated with decreased quality of life and

survival.13, 17, 107, 108 Jones et al. reported that peak VO2 was 27% lower in breast cancer survivors

compared to age and sex-matched sedentary predicted values.13 Moreover, nearly one-third of

survivors had a peak VO2 below the threshold of independent living.13 The marked exercise

intolerance has been linked to a reduced peak exercise cardiac output secondary to a reduced

stroke volume, as peak heart rate (HR) was not different between breast cancer survivors and

controls.17

It is currently not known whether women with reduced exercise capacity are predisposed to

breast cancer or whether exercise intolerance is acquired as a result of cancer therapy. There is a

paucity of studies that have measured peak VO2 in breast cancer patients prior to undergoing

adjuvant chemotherapy. A systematic review by Peel et al. reported that peak VO2 was 17%

lower in breast cancer patients prior to adjuvant therapy compared to age-matched healthy

sedentary predicted values.109 To date, no study has measured exercise cardiac function in breast

cancer patients prior to undergoing chemotherapy. Further, all prior studies have focused

57

primarily on left ventricular (LV) function, therefore uncertainty remains regarding right

ventricular (RV) performance during exercise. Given this uncertainty, the aim of this study was

to test the hypothesis that the impaired peak VO2 in breast cancer patients prior to adjuvant

chemotherapy is attributable to decreased exercise cardiac output despite preserved RV and LV

ejection fraction.

3.1 c) Methods

3.1 c) i: Subjects

Women aged 18 to 70 years with newly diagnosed, histologically confirmed early stage

breast cancer scheduled for anthracycline-based chemotherapy were recruited via collaborating

breast surgeons and oncologists from local hospitals in the Melbourne metropolitan area. Patients

with structural heart disease, sustained cardiac arrhythmias, or a contraindication to cardiac

magnetic resonance imaging were excluded. Age and sex matched control subjects were

recruited from an advertisement to staff of the Baker Heart and Diabetes Institute and the wider

community. Volunteers involved in competitive sport or enrolled in a structured exercise training

program were excluded.

3.1 c) ii: Protocol overview

The study protocol was approved by the Alfred Health Institutional Review Board and

written informed consent was obtained from all subjects. Breast cancer patients performed all

tests prior to anthracycline treatment.

58

3.1 c) iii: Cardiopulmonary Exercise Testing

Incremental exercise testing was performed on an electronically braked upright cycle

ergometer (Lode, Gronigen, Netherlands) with an initial power output of 10-25 watts (W) and

was progressed by 10-30W per minute until volitional exhaustion. Expired gas analysis was

performed using a commercially available metabolic measurement system (True One 2400,

Parvomedics, UT, USA), with the highest value obtained over 30 seconds used as the peak VO2

value. HR was continuously measured (1200W RF Wireless System, Norav, FL, USA) while

blood pressure (Suntech, Tango, NC, USA) was measured every 2 minutes during exercise.

3.1 c) iv: Biochemistry

Peripheral venous blood samples were taken 10 minutes after completion of exercise cardiac

magnetic resonance (cMRI) testing to measure B-type natriuretic peptide (BNP) and troponin I.

Hemoglobin values were obtained from patient medical records.

3.1 c) v: Rest and exercise cardiac magnetic resonance imaging cMRI.

Cardiac images were acquired with a Siemens MAGNETOM Prisma 3T scanner with a 5-

element phased-array coil (Siemens, MAGNETOM Prisma). Ungated, real-time, steady-state

free-precession cine imaging was performed without cardiac/respiratory gating. Forty to 75

consecutive frames were acquired every 36-38 milliseconds at each of 13-18 contiguous 8-mm

slices in the short-axis (SAX) plane, and 50 consecutive frames were acquired at approximately

the same temporal resolution for 11-15 contiguous 8-mm slices in the horizontal long axis (HLA)

plane. Scan duration was approximated to include one full respiratory cycle per slice (~120 and

59

~90 seconds at rest for SAX and HLA planes respectively, ~4-7 heart beats/slice) for gating of

cardiac translation.73

RV and LV volumes were generated by manually tracing the endocardium in the SAX plane

and using the disk summation method.73 To minimize cardiac translation error, contours were

traced in end diastole and end systole for each slice manually gated to respiration. SAX contour

transection points with the HLA plane were displayed for referencing of the atrioventricular

valve plane. Trabeculations and papillary muscles were considered part of the ventricular blood

pools. HR was determined by generating an anatomical M-mode image from the SAX plane

stack. Cardiac index was calculated as the average of RV and LV stroke volume (index to body

surface area) multiplied by HR. Physiologic data was synchronized with images for offline data

analysis. Images were analyzed with RightVol (Right Volume Leuven, Leuven, Belgium), a

MatLab software adjunct.

After resting images were obtained, subjects cycled on a MRI compatible ergometer (MR

Ergometer Pedal, Lode, Groningen, Netherlands) at an intensity equal to 20%, 40% and 60% of

peak power output obtained during the upright incremental cycle exercise test. These workloads

will subsequently be referred to as rest and low, moderate, and peak intensity. Each stage of

exercise was maintained for ≈1 to 3 minutes; 30 seconds to 1 minute to achieve a physiological

steady-state and 1 to 2 minutes for image acquisition. We previously determined that 66% of the

peak power during upright cycling corresponded to peak exercise in a supine position.86

3.1 c) vi: Statistical Analysis

60

Comparison between groups for continuous variables was assessed using student t tests.

Pearson correlation was used to determine linear correlations between variables. Two-Way

ANOVA with post hoc Bonferroni testing was used to compare cardiac volumes and ejection

fraction at different exercise intensities. Unless otherwise stated, data are presented as mean

(standard deviation). A two-sided p value <0.05 was determined as significant. All statistical

analysis were performed with SPSS (version 24 IMB SPSS Statistics, Armonk, NY, USA)

statistics software.

3.1 d) Results

3.1 d) i: Participant characteristics

Twenty-nine women with early breast cancer and 10 age and sex-matched healthy

controls agreed to participate in this study. No significant difference was found between breast

cancer patients and healthy controls for age, height, weight, body mass index, body surface area,

hemoglobin, brain natriuretic peptide, or troponin I. However, body mass index tended to be

higher in the breast cancer group, as fewer healthy control subjects were overweight, and none

were obese (Table 1). Fourteen and fifteen breast cancer patients were scheduled to receive

anthracyclines in a neoadjuvant and adjuvant setting respectively.

Cardiopulmonary exercise performance during upright cycle exercise

Peak exercise power output and VO2 were significantly lower in breast cancer patients

compared to healthy controls (38 21% and 29 17% lower respectively, Table 2). Peak

exercise systolic and diastolic blood pressure and HR were not significantly different between

groups. A sensitivity analysis was conducted to address the potential confounder of surgery prior

61

peak VO2 testing. Fifteen breast cancer patients who had undergone breast surgery (local

resection or mastectomy, scheduled for adjuvant chemotherapy) were compared with the 14

patients planned for surgery after chemotherapy (neo-adjuvant treatment strategy). No significant

difference was found between these groups for age, BMI, BSA, peak power output, VO2 (L/min

or ml/kg/min) or cardiac output (Data supplement).

3.1 d) ii: Cardiac volumes and index at rest, sub-maximal and peak supine

cycle exercise

Complete cMRI data was obtained in 26 of 29 breast cancer patients (one subject withdrew

from the study prior to cMRI testing, HR could not be measured accurately during peak exercise

in one subject- volumes, but not peak cardiac output, are included- one image set was excluded

due to artifact) and 10 healthy controls.

No significant difference was found between groups for rest, sub-maximal and maximal RV

and LV ejection fraction. A significant main group effect was found for RV and LV end-diastolic

volume index, end-systolic volume index, stroke index and cardiac index with values being

lower in breast cancer patients compared to controls (Figures 1 to 3). A significant main

(exercise) intensity effect was found for RV and LV stroke index and ejection fraction being

higher while RV and LV end-systolic volume index were lower during exercise compared to rest

in both breast cancer and controls (Figures 1-3). A significant group by intensity interaction was

found for cardiac index with breast cancer patients having lower values during sub-maximal and

peak exercise compared to controls. Finally, a significant positive relationship was found

between peak VO2 and cardiac output (Figure 4).

62

3.1 e) Discussion

Consistent with limited prior data, we confirmed that, relative to healthy age-matched

controls, women with breast cancer had reduced exercise capacity prior to cancer therapy. We

extend prior research by examining the cardiac mechanisms underpinning this exercise

impairment by comparing changes in RV and LV volumes measured during peak supine cycle

exercise. The principal new finding was that the reduced peak VO2 in breast cancer patients

versus healthy controls was due, in part, to a reduced maximal cardiac index. In turn, the reduced

peak exercise cardiac index was due to a reduced RV and LV stroke index and end-diastolic

volume index as no significant difference was found between groups for RV and LV ejection

fraction.

The mechanisms responsible for reduced biventricular end-diastolic volumes prior to

adjuvant chemotherapy were not studied, however, sedentary aging may play a role. Bhella et al.

(2014) have shown that life-long sedentary (exercise <2 times per week) and casual exercisers

(exercise 2-3 times per week) have lower peak oxygen uptake, LV mass and LV distensibility

than those who exercise >3 times per week.110 Despite increased LV stiffness, less active

individuals did not have impaired systolic impairment or abnormalities, closely mirrored by our

findings.110 One plausible explanation for our finding of lower functional capacity in breast

cancer patients is sedentary deconditioning due to reduced mobility and physical activity

following surgery. However, when we compared patients who had undergone surgery versus

those who had not received any treatment (surgery, chemotherapy or radiation), no difference in

peak VO2 was observed. This null finding is not inconsistent with patient surveys; patients were

referred to the study after surgical recovery and had returned to baseline based on subjective self-

report of symptoms. Accordingly, such a substantial decrement in peak VO2 and ventricular end-

63

diastolic volumes are better explained by longer-term reductions in physical activity volume and

intensity.(4)

Given that the breast cancer patients’ in our study had peak VO2 values that were

approximately 30% lower than controls, it is plausible that the reduced biventricular end-

diastolic volume may be due to increased LV stiffness. Furthermore, a cluster of co-morbidities

associated with a sedentary lifestyle are also linked with increased breast cancer incidence and

mortality, it is perhaps reasonable to hypothesize that a lower peak VO2 may be an additional

clinical breast cancer risk factor and be associated with reduced survival after diagnosis.(13)

Moreover, obesity, metabolic syndrome and muscle atrophy have been associated with increased

breast cancer incidence111-114 and it has been hypothesized that this may be due to a mild pro-

inflammatory state and attenuated immune response.113, 115 On the other hand, exercise inhibits

inflammation in adipose tissue and creates unfavorable conditions for cancer,116 a reduction in

breast cancer incidence and improved survival.117, 118 Thus, it is perhaps not entirely unexpected

that patients diagnosed with breast cancer may have, on average, performed less exercise thereby

resulting in reduced cardiac size and lower exercise capacity, as observed in our current study.

In accordance with the ‘multiple hit’ hypothesis, the reduced peak VO2 in breast cancer

survivors is attributed, in part, to the adverse cardiac effects of chemotherapy.5, 17, 119 To date,

only one study has measured VO2 and its determinants during peak exercise in breast cancer

survivors (n=47, mean age: 59 years, mean time post chemotherapy: 38 months) and age-

matched healthy controls (n=11). The main finding was that the decreased peak VO2 (20%) in

breast cancer survivors versus controls was due to a significantly lower peak exercise cardiac

output and stroke volume, as HR and calculated arterial-venous oxygen difference were not

significantly different between groups.17 We extend these findings and show reduced maximal

64

exercise cardiac output and peak VO2 prior to undergoing chemotherapy. Importantly, our

finding that 45% of the variance in peak VO2 was explained by cardiac output (Figure 4)

suggests that non-cardiac, peripheral factors (eg, muscle blood flow and/or O2 extraction) also

play an important role in limiting peak VO2 prior to adjuvant chemotherapy.

3.1 e) i: Clinical Implications

Given that peak VO2 declines by 10% after 12 weeks of chemotherapy (equal to what is

observed after a decade of sedentary aging),120 our finding that breast cancer patients have

marked exercise intolerance and impaired exercise cardiac output (and likely impaired peripheral

determinants) reserve prior to adjuvant chemotherapy, suggests that interventions that attenuate

the decline in peak VO2 may be an important target of therapy. Indeed, exercise training is an

effective therapy to improve peak VO2 during adjuvant chemotherapy;107 however, the optimal

exercise training program and mechanisms responsible for this favorable adaptation remain

uncertain.121

3.1 e) ii: Limitations

A limitation of this study was that peak VO2 testing was performed during upright exercise

while cardiac volumes were measured in the supine position, therefore the mechanisms

underpinning the reduced maximal cardiac output may differ between groups in the upright

position. However, this is unlikely as peak HR was not different between groups during peak

upright exercise. Also, breast cancer patients and healthy controls relied on a decrease in RV and

LV end-systolic volume to increase stroke index and ejection fraction from rest to peak exercise.

Given that breast cancer patients displayed evidence of sedentary aging, we contend that

65

biventricular end-diastolic volume would remain lower during upright exercise given this

remodeling pattern is associated with decreased cardiac distensibility.

3.1 f) Conclusion

Despite preserved rest, sub-maximal and peak exercise RV and LV ejection fraction, breast

cancer patients prior to adjuvant chemotherapy have marked exercise intolerance, related to

decreased maximal cardiac index, secondary to reduced RV and LV end-diastolic volume.

Peripheral, non-cardiac factors may also play a prominent role in limiting exercise tolerance

prior to adjuvant chemotherapy. Accordingly, interventions that improve cardiovascular and

skeletal muscle function, such as exercise training, initiated at the time of diagnosis may

attenuate the decline in peak VO2 that occurs during adjuvant chemotherapy.

66

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72

3.1 h) Figure Legends

3.1 h) i: Figure 1: Left ventricular end-diastolic volume index (A), end-systolic volume index (B), stroke index (C)

and ejection fraction (D) at rest, sub-maximal and maximal supine cycle exercise. *, Significant

main group (breast cancer vs. control) effect; †, Significant main intensity (low, moderate, and

peak exercise vs. rest).

3.1 h) ii: Figure 2: Right ventricular end-diastolic volume index (A), end-systolic volume index (B), stroke index

(C) and ejection fraction (D) at rest, sub-maximal and peak supine cycle exercise. *, Significant

main group (breast cancer vs. control) effect; †, Significant main intensity (low, moderate, and

peak exercise vs. rest).

3.1 h) iii: Figure 3: Heart rate and cardiac index at rest, sub-maximal and peak supine cycle exercise. *, Significant

main group (breast cancer vs. control) effect; †, Significant main intensity (low, moderate, and

peak exercise vs. rest); ‡, Significant group by intensity interaction.

3.1 h) iv: Figure 4: Relationship between peak VO2 (upright cycle exercise) and peak cardiac output (Q, supine

cycle exercise).

73

3.1 i) Tables

3.1 i) i: Table 1 Participant Characteristics.

Table 4: Participant Characteristics

Parameter BC (n=29) HC (n=10) P Value

Age (years)

median [range]

48 (12)

51 [19-68]

48 (12)

52 [30-66]

0.98

Height (cm) 165 (9) 166 (7) 0.67

Weight (Kg) 72 (19) 66 (9) 0.31

Body mass index (kg/m2) 26.7 (6.9) 23.8 (2.1) 0.21

Normal, n (%) 16 (55) 8 (80) -

Overweight, n (%) 9 (31) 2 (20) -

Obese, n (%) 4 (14) - -

Body surface area (m2) 1.77 (0.22) 1.73 (0.15) 0.49

Hemoglobin (g/dl) 13.0 (1.4)

(n=26)

13.2 (0.5) 0.68

BNP 36.9 (33.6)

(n=26)

26.9 (16.5)

(n=9)

0.38

Troponin I 2.9 (1.3)

(n=26)

3.3 (2.9) 0.58

74

3.1 i) ii: Table 2 Cardiopulmonary Exercise Performance during peak Upright Cycle Exercise

Table 5: Cardiopulmonary exercise performance during upright cycle exercise

Parameter Breast Cancer

(n=29)

Healthy Control

(n=10)

p Value

Power output (W) 142 (47) 228 (51) 0.00003

VO2 (l/min)

VO2 (ml/kg/min)

1.7 (0.4)

25 (6)

2.3 (0.5)

35 (6)

0.0013

0.00009

HR (bpm) 174 (13) 169 (16) 0.39

Systolic blood pressure (mmHg)

Diastolic blood pressure (mmHg)

185 (27)

93 (16)

180 (28)

84 (19)

0.83

0.18

75

3.1 j) Figures

3.1 j) i: Figure 1.

Figure 3: Left Ventricular Volumes; rest to peak exercise

Figure 1: Left ventricular end-diastolic volume index (A), end-systolic volume index (B), stroke

index (C) and ejection fraction (D) at rest, sub-maximal and maximal supine cycle exercise. *,

Significant main group (breast cancer vs. control) effect; †, Significant main intensity (low,

moderate, and peak exercise vs. rest).

Peak Peak

Peak Peak

76

3.1 j) ii: Figure 2.

Figure 4: Right Ventricular Volumes; rest to peak exercise

Figure 2: Right ventricular end-diastolic volume index (A), end-systolic volume index (B),

stroke index (C) and ejection fraction (D) at rest, sub-maximal and peak supine cycle exercise. *,

Significant main group (breast cancer vs. control) effect; †, Significant main intensity (low,

moderate, and peak exercise vs. rest).

Peak

Peak Peak

Peak

77

3.1 j) iii: Figure 3.

Figure 5: Heart Rate and Cardiac Index; rest to peak exercise

Figure 3: Heart rate and cardiac index at rest, sub-maximal and peak supine cycle exercise. *,

Significant main group (breast cancer vs. control) effect; †, Significant main intensity (low,

moderate, and peak exercise vs. rest); ‡, Significant group by intensity interaction.

Peak

Peak

78

3.1 j) iv: Figure 4.

Figure 6: Peak Cardiac Output and Peak Oxygen Uptake

Figure 4: Relationship between peak VO2 (upright cycle exercise) and peak cardiac output

indexed to body surface area (supine cycle exercise).

1

2

3

4

9 11 13 15 17 19 21

Peak

VO

2 (l

/min

)

Peak Q (l/min)

BC

HC r2=0.45

4.1 Leg blood flow is preserved during small muscle mass exercise in long-term

survivors of anthracycline treated breast cancer.

80

4.1 a) Abstract: Background: Persistent anthracycline mediated cardiovascular damage to the periphery may

contribute to reduced exercise tolerance in women with a history of breast cancer (BC).

Methods: We evaluated the hemodynamic responses during submaximal single leg knee

extension (SLKE) exercise in 14 BC survivors (mean age: 61 ± 7 years; mean time post-

anthracycline therapy: 12 ± 6 years) and 9 age and sex-matched control (CON) subjects. Heart

rate (lead II), blood pressure (finger cuff pressure), leg (femoral artery) blood flow (Doppler

ultrasound), vascular conductance and pressure-strain were measured at rest, 25%, 50% and 75%

of maximal power output. Quadriceps mass was estimated from thigh volume and skinfold

measures.

Results: Estimated quadriceps mass was significantly lower in BC survivors compared to CON

(1803 ± 607 vs. 2601 ± 1101 g, p=0.04). No significant difference was found between groups for

maximal SLKE power output (28 ± 11 vs. 34 ± 17 Watts, p=0.33), heart rate (109 ± 14 vs. 103 ±

13 bpm, p=0.36) or mean arterial pressure (122 ± 18 vs. 119 ± 26 mmHg, p=0.33). Rest and sub-

maximal exercise mean arterial pressure, leg blood flow, leg conductance, pressure-strain were

not significantly different between groups.

Conclusion: Resting and sub-maximal exercise leg blood flow, conductance, and femoral artery

stiffness (pressure-strain) are not impaired in long-term survivors of anthracycline treated BC.

Increasing leg muscle mass may be an important target of interventions aimed at improving

cardiovascular health in BC survivors.

81

4.1 b) Introduction Advances in prevention, early detection and therapy have contributed to reduced breast

cancer (BC) mortality.122 As a result of improved survivorship, comorbid conditions compete

with BC as the leading cause of mortality.105 Specifically, BC survivors are at increased risk of

cardiovascular disease and heart failure that is attributed, in part, to the detrimental

cardiovascular effects of anthracycline therapy, including dose-limiting cardiotoxicity.6, 7

Cardiorespiratory fitness, measured as peak oxygen uptake (peak VO2) is the strongest

predictor of cardiovascular and all-cause mortality in healthy and clinical populations, and is

reduced in BC survivors by 20%.13, 15, 123, 124 While reduced peak VO2 is associated with reduced

peak cardiac output in BC, emerging evidence suggests that non-cardiac ‘peripheral’

mechanisms may also contribute to reduced exercise tolerance.16, 17, 19, 123, 124 Indeed, Howden et

al. recently reported that the reduced peak VO2 (15%) from pre to post-anthracycline therapy was

due to a decline in arterial-venous oxygen difference, as peak cardiac output did not change

during this time. Moreover, Chaosuwannakit et al. demonstrated that compared to baseline,

resting aortic distensibility decreased (54%) and aortic pulse wave velocity increased (95%) after

of 4-months of anthracycline chemotherapy in 40 cancer patients (BC, n=19) with no change in

age-and sex-matched controls.35 Mizia-Stec et al. extended these findings by demonstrating that

carotid artery stiffness increased from pre-to post anthracycline therapy in 35 BC patients.34

Finally, Didier and colleagues comparing cancer survivors (n=11, BC n=10, anthracycline

treated n=3) and age- and sex-matched controls (n=9) found that the former group had an

impaired muscle blood flow response to sub-maximal (20% of maximal voluntary contraction)

rhythmic hand-grip exercise.37

82

Taken together, the few studies performed to date suggest that vascular stiffening and

reduced muscle blood flow may contribute to the impaired exercise tolerance found in cancer

survivors. Currently, no study has examined whether blood flow to major locomotor muscle is

impaired, and if this impairment is related to arterial stiffening in BC survivors. Single leg knee

extension (SLKE) where the limiting role of the heart is minimized, has been used to identify

peripheral blood flow limitations to exercise in non-cancer populations (heart failure patients

with preserved or reduced ejection fraction) who have abnormal exercise cardiac function.125-127

Using SLKE exercise we tested the hypothesis that leg blood flow and vascular conductance

would be significantly reduced, and femoral artery stiffness would be significantly increased in

anthracycline treated BC survivors compared to age-and sex-matched controls (CON).

4.1 c) Methods:

4.1 c) i: Participants: Female BC survivors and CON were recruited from the local Dallas/Fort Worth community

via recruitment flyers and by word of mouth. Informed, written consent was obtained from all

subjects, and the study was approved by the University of Texas at Arlington Institutional

Review Board.

Breast cancer survivors had to have completed anthracycline chemotherapy more than 1 year

prior to study enrollment. Exclusion criteria for both groups were: orthopedic limitation to

exercise, resting blood pressure exceeding 160/100 mmHg for BC survivors or 140/90 mmHg for

CON, and inability to acquire a femoral artery ultrasound image of sufficient quality for analysis.

Additional exclusion criteria for control participants were presence of cardiovascular disease or

current cardio-active medication.

83

4.1 c) ii: Protocol Overview: Visit One: Physical activity and leg muscle mass: Participants self-reported their level of

physical activity (Stanford Leisure Time Activity Category Item) and medical history using a

researcher developed questionnaire.128 Height and weight were measured using a standiometer

and electronic scale, and thigh length, circumference (proximal, mid and distal) and skinfold

were measured using a flexible measuring tape and skinfold calipers to estimate quadriceps

muscle mass using a previously validated equation by Andersen and Saltin.129

Incremental SLKE exercise test: Participants were seated on a custom SLKE ergometer during

which time a finger blood pressure cuff (Finapress, AD Instruments, Australia) and ECG (lead

II) were continuously measured. An image of the right, common femoral artery was optimized

(landmarked at the femoral artery bifurication) using B-mode ultrasound. After optimization, a

duplex image was acquired, with the pulsed wave velocity sample volume positioned 2 cm

proximal to the femoral artery bifurication. The angle of insonation was limited to 60ᵒ, and the

sample volume was parallel to the artery walls. Two-minutes of resting duplex ultrasound were

recorded using a video capture device (Video Capture, Elgato, Germany) for offline analysis.

Participants were then familiarized with SLKE exercise. Specifically, they were instructed to

“kick” to a metronome set at 50 beats per minute, and to relax their leg entirely during rebound

driven by flywheel momentum. Once participants achieved a cadence of 50 revolutions per

minute, the resistance was increased to 10 watts (W) and then increased by 10 W every 2

minutes thereafter until volitional exhaustion or an inability to adhere to the pre-specified

cadence.

Visit Two: Peripheral hemodynamic responses during sub-maximal SLKE exercise: Participants

were instrumented as per Visit 1. After image optimization, 2 minutes of duplex ultrasound were

84

recorded, and then participants began their first sub-maximal SLKE power output. Workloads

were targeted to 25, 50 and 75% of the previously determined maximal power output. Each

workload was performed for 5 minutes with concomitant femoral artery imaging. Five minutes

of post-exercise recovery were recorded. Workloads were separated by 10 minutes of rest. In a

subset of participants, a third visit identical to Visit 2, was performed to test repeatability. The

average measures interclass correlation was 0.92 for femoral artery blood flow. Data from Visit 2

are reported.

4.1 c) iii: Measurements: Duplex ultrasound recordings were analyzed offline using automated edge detection

software (CardioSuite, Quipu, Italy). Femoral artery blood flow was calculated as: π(Dmean/2)2 x

V/60 when Dmean = femoral artery diameter in cm, V/60 = blood velocity in cm/min. Blood flow

during SLKE was calculated using a noise-free 30-60 second velocity average from the final 120

seconds of exercise (stable, steady state), and a 60 second diameter average from post-exercise

recovery. Data were cleaned to remove non-physiologic data-points and blood flow was

normalized to estimated quadriceps mass. Single leg vascular conductance was calculated as: leg

blood flow/mean arterial pressure where mean arterial pressure was averaged over the final

minute of each stage. Femoral artery stiffness was quantified as pressure-strain (Young’s elastic

modulus, E = K[pulse pressure]/strain) where strain = [Ddiastolic – Dsystolic]/Ddiastolic and K = 133.3

N/m2/mmHg, as previously described.130 Femoral artery systolic and diastolic diameters were

measured immediately upon exercise cessation (<20 seconds) using 3 independent cardiac cycles

within a 10 second window while pulse pressure was averaged over the same period.

4.1 c) iv: Statistical analysis: Continuous descriptive and outcome variables were compared between groups using

independent t-tests. In the case of non-parametric distribution of continuous variables (confirmed

85

by the Shapiro-Wilk test) or scale measures, the Mann Whitney U-test was used. A 2-way

repeated measures ANOVA was used to compare blood flow, pressure, conductance, heart rate,

and pressure-strain responses to SLKE. A Pearson’s correlation was used to test for a bivariate

relationship between femoral artery stiffness and blood flow. Data are presented as mean ±

standard deviation, significance was set at p<0.05.

4.1 d) Results:

4.1 d) i: Participants: Fifteen BC survivors and 11 age- and sex-matched CON were recruited to the study. One

participant from each group was excluded prior to Visit 2 with the post-hoc exclusion criterion of

the presence of an arterial branch superior to the femoral artery bifurcation resulting in an

atypical femoral artery blood velocity profile. One CON participant was excluded due to high

blood pressure. The BC survivors were on average 12 ± 6 years post anthracycline therapy, and

93% of participants received doxorubicin while 71% underwent radiation therapy and hormone

therapy each (Table 1). The BC and CON were well matched for age, body mass index and self-

reported physical activity. The BC survivors had a significantly lower estimated quadriceps mass

(1803 ± 607 vs. 2601 ± 1002 g, p=0.04) and quadriceps mass as a percent of body mass (2.6 ±

0.7 vs 3.6 ± 1.1 %, p=0.02) compared to CON.

4.1 d) ii: Incremental to maximal SLKE Exercise: Power output (BC: 28 ± 11 vs. CON: 34±17 W, p=0.35), heart rate (BC: 109 ± 14 vs. CON:

103 ± 13 bpm, p=0.36) and mean arterial pressure (BC: 122 ± 18 vs. CON: 119 ± 26 mmHg,

p=0.33) were not different between groups during maximal SLKE exercise (Figure 1, table 2).

4.1 d) iii: Peripheral hemodynamic at rest and sub-maximal SLKE Exercise: Resting heart rate, mean arterial pressure, leg blood flow and vascular conductance were

similar between BC and CON (Figure 1). Due to video-capture malfunction, duplex ultrasound

86

records were lost at the 50 and 75% submaximal workloads in one CON participant. As maximal

power was not different between groups, relative work rates at 25, 50 and 75% of maximal

SLKE exercise were similar between groups. Additionally, heart rate, mean arterial pressure and

leg vascular conductance were similar at rest and during submaximal exercise (Figure 1).

Femoral artery blood flow was not significantly different between groups at rest or during

submaximal exercise (Figure 1). Similarly, no group differences were found in femoral artery

strain (Wilks’ Lambda, p=0,216 for intensity effect) or pressure-strain at rest and immediately

following sub-maximal exercise (Figure 2). Leg blood flow, conductance, mean arterial pressure

and pressure-strain increased from rest to submaximal exercise (Figures 1, 2).

4.1 d) iv: Relationship between femoral artery stiffness and blood flow: No relationship was found between femoral artery blood flow and femoral artery stiffness as

measured as pressure-strain at rest, or immediately after 25, 50 or 75% of maximal SLKE (Table

3).

4.1 e) Discussion Breast cancer survivors have severely reduced exercise tolerance.109 Emerging evidence

suggests that anthracycline chemotherapy may be associated with peripheral dysfunction that

may contribute to decreased exercise tolerance.13, 17 The major new finding of this study was that

leg blood flow and conductance during sub-maximal SLKE exercise, where the limiting role of

the heart is minimized, was not significantly different between BC and age and sex-matched

CON. Further, to our knowledge we provide the first data on femoral artery stiffness at rest and

immediately following submaximal exercise in long-term survivors of anthracycline treated BC

and report no difference for this outcome between BC and CON.

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4.1 e) i: Leg Blood Flow in Long-Term BC Survivors: Our finding of preserved leg blood flow during non-cardiac limited exercise is contrary to

findings from Didier et al. who reported that forearm blood flow was attenuated in 11 long-term

cancer survivors treated with adjuvant therapy (n=10 BC, mean age=56, mean 3 years post-

therapy).37 The attenuated blood flow was attributed to a blunted arterial blood pressure

response, secondary to impaired left ventricular ejection time index derived from an arterial

pressure waveform. In contrast, we found no differences in the heart rate or blood pressure

responses to exercise, and found a comparable blood flow response when indexed to 100 g of

muscle mass during moderate intensity exercise (25 and 50% of peak SLKE, figure 1). Further,

at the highest exercise intensity (75% of peak SLKE) we also did not unmask a leg blood flow

deficit in BC. The discrepancy in findings may be due to regional differences in muscle mass and

adiposity. Critically, we measured blood flow at the level of a conduit artery which supplies the

entire leg. Physiologic increases in blood flow should closely match metabolic demand;

however, blood may be delivered to vascular beds in close proximity resulting in normal

“muscle” blood flow. Metabolite spillover, conductive vasodilation and heat cause increased

blood flow to local tissues, including to adipose tissue adjacent to active muscle.131 Thus, given

that we observed reduced quadriceps mass but preserved leg conductance in BC compared to

controls, it is possible that blood flow was shunted through non-force producing tissue. In the

forearm, this effect may be minimized due to smaller active muscle mass, lower vascular

conductance, lower adiposity, and lower work rates producing fewer metabolites and less heat

spill over to non-force producing tissue.132

A consequence of blood flow shunting through less metabolically active tissue (inactive

muscle, adipose tissue, skin) is reduced oxygen uptake during whole body peak exercise. In heart

failure with preserved ejection fraction, a syndrome marked by extreme exercise intolerance, the

88

ratio of intermuscular fat to skeletal muscle is inversely related to peak oxygen uptake.30 This

physiologic relationship is explained by shunting of blood through intermuscular fat, resulting in

an impaired matching of cardiac output and peak VO2. In a study of 14 cancer survivors (mean

age 54 years, >12-months post-therapy) and 14 age- and sex-matched controls, the same effect

was observed whereby intermuscular fat was associated with reduced exercise capacity.29

Our findings demonstrate preserved leg blood flow in response to SLKE despite reduced

quadriceps mass in anthracycline treated BC survivors. Preserving or increasing muscle mass is

therefore an important target for interventions directed at increasing exercise tolerance in BC.

Dietary and exercise interventions have proven efficacy for reducing adiposity and preserving or

increasing muscle mass.133-136 Accordingly, favorable changes in body composition could

dramatically improve cardiorespiratory fitness through increasing efficiency of blood flow

delivery to active muscle, thus preventing progression of BC survivors towards a HFpEF-like

phenotype.

4.1 e) ii: Femoral Artery Stiffness in Long-Term BC Survivorship Vascular toxicity, specifically vascular stiffening, is a proposed mechanism of anthracycline

related decline in cardiovascular health.6 To our knowledge, only two studies have measured

conduit artery stiffness. Mizia-Stec et al. reported a decrease in common carotid artery

compliance from pre to post-anthracycline therapy (n=35, age: 50 years)34 while Koelwyn et al.

report no difference in common carotid artery compliance or distensibility (n=30, 6 years post

treatment, age: 61 years) compared to control subjects.19 Koelwyn et al. also reported impaired

ventricular-arterial coupling in anthracycline treated BC survivors under stress conditions (25, 50

and 75% of peak cycling work rate). Arterial elastance, estimated from echocardiography

derived cardiac volumes and brachial artery end-systolic blood pressures, was not impaired in

89

BC. Here, we measured arterial stiffness in a large conduit artery feeding major locomotor

muscle, and found no decrement in femoral artery pressure-strain. Importantly, pressure strain

was not impaired at rest, nor under conditions of increasing blood flow or pressure- consistent

with estimated arterial elastance as previously reported.19 Finally, we found no relationship

between leg blood flow and femoral artery pressure-strain (table 3). Therefore, stiffening of

conduit arteries does not appear to limit blood flow to major locomotor muscle during sub-

maximal SLKE where the limiting role of the heart is minimized.

4.1 e) iii: Limitations: A limitation of the study is that blood flow was measured in the common femoral artery,

rather than at the level of active muscle. While we have interpreted our findings in the context of

the available literature, it is unclear whether perturbations in blood flow delivery to active

muscle microvasculature exist, or whether muscle oxygen utilization is preserved in long term

survivors of anthracycline treated BC. To our knowledge, no data on mitochondrial function are

available, nor on active-muscle blood flow and diffusive oxygen conductance- two possible

contributors to exercise intolerance and functional disability.

4.1 e) iv: Conclusion: Leg blood flow, vascular conductance and arterial stiffness during sub-maximal SLKE

exercise are not significantly different between BC survivors treated with anthracycline

chemotherapy and age and sex-matched CON. Body composition (i.e. increasing lower extremity

muscle mass) may be an important target when tailoring dietary and exercise interventions to

improve cardiovascular health in long-term survivors of anthracycline treated BC.

90

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and killing the heart: A novel model to explain elevated cardiovascular disease and mortality risk among

women with early stage breast cancer. Prog Cardiovasc Dis. 2019.

7. McGowan JV, Chung R, Maulik A, Piotrowska I, Walker JM and Yellon DM. Anthracycline

Chemotherapy and Cardiotoxicity. Cardiovascular Drugs and Therapy. 2017;31:63-75.

13. Jones LW, Courneya KS, Mackey JR, Muss HB, Pituskin EN, Scott JM, Hornsby WE, Coan AD,

Herndon JE, Douglas PS and Haykowsky M. Cardiopulmonary Function and Age-Related Decline

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2012;30:2530-2537.

15. Gulati M, Pandey DK, Arnsdorf MF, Lauderdale DS, Thisted RA, Wicklund RH, Al-Hani AJ and

Black HR. Exercise capacity and the risk of death in women - The St James Women Take Heart Project.

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16. Khouri MG, Hornsby WE, Risum N, Velazquez EJ, Thomas S, Lane A, Scott JM, Koelwyn GJ,

Herndon JE, Mackey JR, Douglas PS and Jones LW. Utility of 3-dimensional echocardiography, global

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17. Jones LW, Haykowsky M, Pituskin EN, Jendzjowsky NG, Tomczak CR, Haennel RG and

Mackey JR. Cardiovascular reserve and risk profile of postmenopausal women after chemoendocrine

therapy for hormone receptor - Positive operable breast cancer. ONCOLOGIST. 2007;12:1156-1164.

19. Koelwyn GJ, Lewis NC, Ellard SL, Jones LW, Gelinas JC, Rolf JD, Melzer B, Thomas SM,

Douglas PS, Khouri MG and Eves ND. Ventricular-Arterial Coupling in Breast Cancer Patients After

Treatment With Anthracycline-Containing Adjuvant Chemotherapy. ONCOLOGIST. 2016;21:141-149.

29. Reding KW, Brubaker P, D’Agostino R, Kitzman DW, Nicklas B, Langford D, Grodesky M and

Hundley WG. Increased skeletal intermuscular fat is associated with reduced exercise capacity in cancer

survivors: a cross-sectional study. Cardio-Oncology. 2019;5:3.

30. Haykowsky MJ, Kouba EJ, Brubaker PH, Nicklas BJ, Eggebeen J and Kitzman DW. Skeletal

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preserved ejection fraction. Am J Cardiol. 2014;113:1211-6.

34. Mizia-Stec K, Goscinska A, Mizia M, Haberka M, Chmiel A, Poborski W and Gasior Z.

Anthracycline chemotherapy impairs the structure and diastolic function of the left ventricle and induces

negative arterial remodelling. Kardiol Pol. 2013;71:681-90.

35. Chaosuwannakit N, D'Agostino R, Hamilton CA, Lane KS, Ntim WO, Lawrence J, Melin SA,

Ellis LR, Torti FM, Little WC and Hundley WG. Aortic Stiffness Increases Upon Receipt of

Anthracycline Chemotherapy. JOURNAL OF CLINICAL ONCOLOGY. 2010;28:166-172.

37. Didier KD. Altered Blood Flow Response to Small Muscle Mass Exercise in Cancer Survivors

Treated With Adjuvant Therapy. 2017;6.

105. Patnaik JL, Byers T, DiGuiseppi C, Dabelea D and Denberg TD. Cardiovascular disease

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a retrospective cohort study. BREAST CANCER RESEARCH. 2011;13:R64.

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123. Foulkes S, Howden EJ, Bigaran A, Janssens K, Antill Y, Loi S, Claus P, Haykowsky MJ, Daly

RM, Fraser SF and La Gerche A. Persistent Impairment in Cardiopulmonary Fitness following Breast

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124. Beaudry R, Howden, E., Foulkes, S., Bigaran, A., Claus, P., Haykowsky, M., La Gerche, A.

Determinants of Exercise Intolerance in Breast Cancer Patients Prior to Anthracycline Chemotherapy.

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126. Barrett-O'Keefe Z, Lee JF, Berbert A, Witman MA, Nativi-Nicolau J, Stehlik J, Richardson RS

and Wray DW. Hemodynamic responses to small muscle mass exercise in heart failure patients with

reduced ejection fraction. Am J Physiol Heart Circ Physiol. 2014;307:H1512-20.

127. Magnusson G, Gordon A, Kaijser L, Sylven C, Isberg B, Karpakka J and Saltin B. High intensity

knee extensor training, in patients with chronic heart failure. Major skeletal muscle improvement. Eur

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128. Kiernan M, Schoffman DE, Lee K, Brown SD, Fair JM, Perri MG and Haskell WL. The Stanford

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130. Godia EC, Madhok R, Pittman J, Trocio S, Ramas R, Cabral D, Sacco RL and Rundek T. Carotid

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135. Murphy JC, McDaniel JL, Mora K, Villareal DT, Fontana L and Weiss EP. Preferential

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4.1 g) Figure Legends:

4.1 g) i: Figure 1: Single leg knee extension power (A), and blood flow (B), mean arterial pressure (C), leg

conductance (D) and heart rate (E) responses. Significant intensity effect (p<0.05) indicated by

*vs rest, † vs 25%, ‡ vs 50%, # vs 75%.

4.1 g) ii: Figure 2: Femoral artery pressure-strain at rest and during submaximal exercise. Significant intensity

effect (p<0.05) indicated by *vs rest, † vs 25%.

93

4.1 h) Tables:

4.1 h) i: Table 1: Participant characteristics. Table 6 Participant Characteristics

Parameter BC (n=14) Control (n=9) P

Age (years) 61 (7) 59 (7) 0.38

BMI (kg/m²) 26.2 (3.8) 27.9 (4.7) 0.54

Estimated Quadriceps Mass (g) 1803 (607) 2601 (1102) 0.04

Quadriceps % of Body Mass 2.6 (0.7) 3.6 (1.1) 0.02

LTAC Score (1-6) 3 (1) 3 (1) 0.88*

Comorbidities (n, %)

Arthritis 5 (36) 0 (0)

Asthma 2 (14) 1 (11)

Hypothyroid 1 (7) 2 (25)

High Cholesterol 4 (29) 1 (11)

Hypertension 2 (14) 0 (0)

Breast Cancer

Stage (n, 0/I/II/III) 1/3/7/3 -

Doxorubicin/Epirubicin (n) 13/1 -

Time post therapy (years) 12 (6) -

Surgery

(Lumpectomy/Mastectomy)

5/9 -

Radiation Therapy (n) 10 -

Biologic Therapy (n) 3 -

Hormone Therapy (n) 10 -

L-TAC: Stanford Leisure Time Activity Category Item. *Mann-Whitney U test.

94

4.1 h) ii: Table 2: Power output, heart rate and mean arterial pressure during

maximal SLKE exercise. Table 7 Maximal single leg knee extension parameters

Parameter BC

(n=14)

Control

(n=9)

P

Power (W) 28 (11) 34 (17) 0.35

Heart Rate (bpm) 109 (14) 103 (13) 0.36

Mean Arterial Pressure

(mmHg)

122 (18) 119 (26) 0.33

95

4.1 h) iii: Table 3: Pearson’s correlations between femoral artery blood flow

and pressure-strain at rest and immediately after submaximal SLKE exercise. Table 8 Pearson's correlations between femoral artery blood flow and pressure-strain

Blood Flow and

Pressure-Strain

Bivariate

Relationship

Combined Pearson’s R

(p)

BC Only Pearson’s

R (p)

Rest -0.089 (0.688) -0.157 (0.592)

25% SLKE 0.150 (0.495) -0.043 (0.883)

50% SLKE -0.142 (0.528) -0.203 (0.487)

75% SLKE -0.253 (0.255) -0.312 (0.278)

96

4.1 i) Figures:

4.1 i) i: Figure 1: SLKE power and hemodynamic responses to SLKE exercise.

Single leg knee extension power (A), and blood flow (B), mean arterial pressure (C), leg

conductance (D) and heart rate (E) responses. Significant intensity effect (p<0.05) indicated by

*vs rest, † vs 25%, ‡ vs 50%, # vs 75%.

Figure 7Power output, leg blood flow, mean arterial pressure and leg conductance at rest and during single leg knee extension

97

4.1 i) ii: Figure 2: Femoral artery pressure-strain at rest and immediately after

submaximal SLKE exercise

;

Significant intensity effect (p<0.05) indicated by *vs rest, † vs 25%.

Figure 8 Femoral artery pressure-strain

0

1000

2000

3000

4000

5000

0 25 50 75

Pre

ssu

re-S

trai

n (

N/m

²)

Work Rate (% Max)

98

99

5.1 Exercise intolerance in anthracycline-treated breast cancer survivors: the role

of skeletal muscle bioenergetics, oxygenation and composition.

Beaudry RI1, Kirkham AA2, Thompson RB2, Grenier JG2, Mackey JR3, Haykowsky MJ1.

1College of Nursing and Health Innovation, University of Texas at Arlington, Arlington, USA

2Department of Biomedical Engineering, University of Alberta, Edmonton, Canada

3Department of Oncology, University of Alberta, Edmonton, Canada

Submitted to The Oncologist

100

5.1 a) Abstract Background: Peak oxygen consumption (VO2) is reduced in women with a history of breast

cancer (BC). We measured leg blood flow, oxygenation, bioenergetics, and muscle composition

in women with BC treated with anthracycline chemotherapy (n=16, mean age: 56) and age- and

body mass index-matched controls (n=16).

Methods: Whole-body peak VO2 was measured during cycle exercise. 31Phospohous magnetic

resonance (MR) spectroscopy was used to measure muscle bioenergetics during and after

incremental to maximal plantar flexion exercise (PFE). MR imaging was used to measure lower

leg blood flow, venous saturation (SvO2) and VO2 during submaximal PFE, and abdominal,

thigh and lower leg intermuscular fat (IMF) and skeletal muscle (SM).

Results: Whole body peak VO2 was significantly lower in BC survivors vs. controls (23.1±7.5

vs. 29.5±7.7 ml/kg/min). Muscle bioenergetics and mitochondrial oxidative capacity were not

different between groups. No group differences were found during submaximal PFE for lower

leg blood flow, SvO2 or VO2. The IMF:SM ratio was higher in the thigh and lower leg in BC

survivors (0.36±0.19 vs 0.22±0.07, p=0.01; 0.10±0.06 vs 0.06±0.02, p=0.03, respectively) and

were inversely related to whole-body peak VO2 (r= -0.71, p=0.002; r=-0.68, p=0.003,

respectively). In the lower leg, IMF:SM ratio was inversely related to VO2 and O2 extraction

during PFE.

Conclusion: SM bioenergetics and oxidative capacity in response to PFE are not impaired

following anthracycline treatment. Abnormal SM composition (increased thigh and lower leg

IMF:SM ratio) may be an important contributor to reduced peak VO2 during whole body

exercise among anthracycline-treated BC survivors.

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5.1 b) Implications for Practice: Peak oxygen consumption (VO2) is reduced in breast cancer (BC) survivors and is prognostic of

increased risk of cardiovascular disease-related and all-cause mortality. We demonstrated that in

the presence of deficits in peak VO2 one year post-anthracycline therapy, skeletal muscle

bioenergetics and oxygenation are not impaired. Rather, body composition deterioration (e.g.

increased ratio of intermuscular fat to skeletal muscle) may contribute to reduced exercise

tolerance in anthracycline BC survivors. This finding points to the importance of lifestyle

interventions including caloric restriction and exercise training to restore body composition and

cardiovascular health in the BC survivorship setting.

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5.1 c) Introduction Advances in prevention, early detection, and treatment of early stage breast cancer (BC) have

dramatically improved survival, and consequently, other conditions including cardiovascular

disease (CVD) now compete with BC as the primary cause of mortality.1, 2 Women with a history

of BC are at greater risk for CVD and related mortality relative to women without BC. It is well

established that following treatment for BC, cardiorespiratory fitness, measured as peak oxygen

uptake (peak VO2) during whole-body exercise, is ≈20% lower than age- and sex-matched

controls.13, 16, 17, 19, 124, 137 Low cardiorespiratory fitness is associated with increased heart failure

incidence, cardiovascular events, and both CVD disease-related and all-cause mortality.138

Several BC therapies can cause cardiovascular injury, including anthracycline chemotherapy,

which is associated with a dose-dependent, progressive, myocardial injury.7 Accordingly,

impairments in peak VO2 could be attributed to ‘central’ limitations that reduce peak exercise

cardiac output.17, 123 However, ‘peripheral’ impairments in exercise blood flow, citrate synthase

activity, and relative reductions in oxidative muscle fiber and capillarity have been reported in

individuals after systemic cancer therapy.31, 139 Abnormalities in skeletal muscle composition may

also limit exercise tolerance; Reding et al. reported peak VO2 was inversely related to the ratio of

intermuscular fat (IMF) to skeletal muscle (SM) within the paraspinal muscles in cancer

survivors.29 Given that O2 consumption during cycle exercise occurs predominantly in the muscles

of the thigh and lower leg, skeletal muscle composition in the leg is likely a more important

determinant of peak VO2. Prior reports studied populations heterogeneous for both cancer type

and treatment, or only assessed acute changes during active treatment. Accordingly, the aims of

this study were to: 1) comprehensively compare non-invasive measures of leg SM composition,

rest and exercise blood flow, O2 extraction, VO2 and oxidative metabolism during plantar flexion

exercise (PFE) using magnetic resonance (MR) imaging, 31P spectroscopy and whole body (cycle)

103

peak VO2 in BC survivors approximately one-year post-anthracycline treatment and age- and BMI-

matched non-cancer controls; and 2) evaluate the relationship between thigh, lower leg, and

paraspinal SM composition and whole-body peak VO2. We hypothesized that BC survivors would

have attenuated exercise leg blood flow, impaired SM oxidative capacity, and unfavorable SM

composition compared to controls, and that IMF:SM ratio in the leg would be inversely related to

peak VO2. An exploratory objective was to assess underlying effects of the IMF:SM ratio by

examining its relationship with measures of exercise metabolism, blood flow, VO2, and O2

extraction in the lower leg of anthracycline-treated BC survivors and non-cancer controls.

5.1 d) Methods

5.1 d) i: Participants Thirty-two women participated in this cross-sectional study, with sixteen BC survivors and

sixteen age and BMI matched non-cancer controls. The women with BC were recruited from a

randomly selected cohort of 75 BC survivors who received anthracycline treatment at the Cross

Cancer Institute (Edmonton, Canada) in the previous year, by mailing an invitation to participate.

The women with BC who responded to the letter were then further screened for eligibility,

including an additional inclusion criterion of a minimum of 3.5 months since receipt of their last

anthracycline-based chemotherapy treatment to avoid effects of acute cardiotoxicity.11 The

control participants were recruited from the local community via word of mouth and recruitment

posters. Exclusion criteria for both groups included a history of cardiovascular disease, diabetes,

or lung disease, and contraindications to MRI. Controls were also excluded if they had a history

of cancer of any type. Informed, written consent was obtained from all participants, and the study

was approved by the University of Alberta Research Ethics Board.

104

5.1 d) ii: Cardiorespiratory fitness Exercise testing was performed on an electrically-braked upright cycle ergometer (Ergoselect

II 1200, Ergoline, Germany) using a staged protocol that started at 20 watts (W) and increased by

15 W every minute until volitional exhaustion. Expired gas was collected and analysed using a

commercially available metabolic measurement system (Encore229 Vmax, SensorMedics, USA)

and the highest 20-second average was used as whole-body peak VO2. Heart rate (lead II ECG)

was monitored continuously while rating of perceived exertion (RPE) was recorded every 2

minutes and at peak exercise.

5.1 e) MR Imaging and 31P Magnetic Resonance Spectroscopy Skeletal muscle function, resting and exercise metabolism, and composition were evaluated

using a 3.0 T MRI system (PRISMA, Siemens Healthineers, Erlangen, Germany).

Incremental PFE – 31P Metabolism

Subjects performed an incremental to maximal, unilateral (left leg), supine PFE test using a

commercially available MRI ergometer (Trispect Module, Ergospect, Austria; Figure 1) inside

the scanner. Plantar flexion exercise utilizes relatively small muscle mass, where the limiting

role of the heart is minimized,140 and elicits a metabolic response similar to the common daily

activity of stair climbing.141 The initial power output was set at 4 W and increased by 2 W every

minute until volitional exhaustion. A metronome provided a consistent cadence of 30

contractions/minute and a study member provided feedback and encouragement from inside the

scanner room to ensure test consistency. Tests were terminated at volitional exhaustion or

technique breakdown (involvement of non-plantar flexor muscle groups or inability to perform

30 contractions/minute).

105

31P spectra, averaged over 12-second intervals, were acquired continuously from rest,

throughout exercise, and for four minutes of recovery after cessation of exercise (TR = 1.5

seconds, flip angle = 30 degrees, 8 cm diameter 31P radiofrequency coil was used for excitation

and signal reception). Relative concentrations (area under the individual spectra) of

phosphocreatine (PCr) and inorganic phosphate (Pi) were measured to generate Pi:PCr ratios,

and intracellular pH was calculated from the chemical shift difference between the Pi and PCr

signals.142 The Pi:PCr ratio and pH were compared at 60% of peak power output as a

representative moderate-intensity workload that occurred prior to a marked reduction in pH for

all participants. From the time of exercise completion, a mono-exponential curve was fit to the

PCr recovery data to calculate the PCr recovery time constant, (PCr). The Pi:PCr ratio at the

moderate exercise intensities and the PCr recovery were measured as indicators of skeletal

muscle oxidative metabolism.143 31P spectra were analyzed using custom, in-house developed

MATLAB code (MATLAB, MathWorks, USA).

Sub-maximal PFE Leg Blood Flow, Oxygen Extraction and VO2

Following 15-20 minutes of recovery after the incremental test, participants performed four

minutes of constant work load sub-maximal PFE using the right leg, at 60% of peak power

output. Blood flow and venous oxygen saturation were measured in the right superficial femoral

vein just superior to the knee at rest (prior to exercise), and immediately (<1 second) after

cessation of exercise, using a previously described method.140, 144-146 Briefly, MR susceptometry-

based oximetry was used to estimate venous oxygen saturation (SvO2) based on the magnetic

field shift associated with the concentration of deoxyhemoglobin.145 The same MRI acquisition

included simultaneous quantification of blood flow (ml/min) in the superficial femoral vein.

Images were acquired perpendicular to the superficial femoral vein (axial image orientation)

106

proximal to the knee (Figure 1). Together with arterial oxygen saturation (SaO2, finger pulse

oximetry) and hemoglobin (Hb) concentration (obtained from blood tests immediately prior to

the MRI scan), lower leg VO2 and O2 extraction were calculated in accordance with the Fick

Principle:

Lower leg VO2 (ml/min) = blood flow (ml/min) · [(SaO2 – SvO2, %) · Hb g/dL] · 1.34 mL O2/g Hb.

While Lower leg O2 extraction was calculated as:

O2 extraction = (SaO2 – SvO2)/SaO2 x 100%.

Skeletal Muscle and Fat Composition

At each of the lower leg, thigh and abdomen regions, a multi-slice multi-echo acquisition was

used to reconstruct water and fat separated images using the modified Dixon approach (Figure

2).147 Typical image parameters include a 4 mm slice thickness, axial slice orientation, 1.0-1.3

mm in-plane resolution, with 30 slices covering the lower leg, 50 slices covering the thigh and 12

slices in the abdomen, with a sub-set of images selected for analysis at each location. For the

lower leg, slices were analysed from 5 cm below the proximal end of tibial plateau, as identified

on a sagittal localizer image of the lower leg, to the distal elimination of the gastrocnemius. For

the thigh, 10 consecutive slices were analyzed starting 40 mm from the most distal point of the

femur, as identified using the sagittal localizer image of femur. In the abdomen, three slices

centered at the middle of the third lumbar vertebrae were identified using sagittal localizer

images of the spine.

Absolute volumes of muscle (SM), intermuscular fat (IMF) and subcutaneous fat were

measured in the lower leg, thigh and abdomen. Visceral fat (VF) was also measured in the

abdomen. For image segmentation, custom MATLAB code was used to perform semi-automated

107

analysis to identify the boundaries of subcutaneous fat, SM, IMF, VF and bones. All regions

were manually checked and adjusted in each slice to ensure accuracy of the automated

delineation. Volumes for each component were quantified using the disk summation method at

each of the three regions (lower leg, thigh and abdomen). The SM and IMF volumes were used

to calculate the ratio of IMF to SM as well as the SM fat fraction (IMF/(IMF+SM)*100%) in

each region.

5.1 e) i: Descriptive information Cardiovascular risk factors and usual physical activity in the past month were collected

by researcher-developed questionnaire and the Godin Leisure-Time Exercise Questionnaire.148

Diagnosis and treatment history were extracted from medical records.

Statistical Analysis

Categorical descriptive characteristics were compared between groups using chi square

tests or Fisher’s exact test for variables with cell size above or below n=5, respectively. Student’s

t-tests for independent samples were used to compare continuous variables between groups. In

the case of non-parametric distribution of continuous variables (confirmed by the Shapiro-Wilk

test), the Mann Whitney U-test was used instead. Bivariate associations between IMF:SM and

SM fat fraction in each region and relative peak VO2 were characterized with Pearson’s product-

moment correlations within both groups combined and confirmed within the BC group alone.

Pearson’s correlations were also used to characterize associations between our parameters of

lower leg function and metabolism and the IMF:SM ratio in the lower leg. Continuous data are

presented as mean ± standard deviation.

108

5.1 f) Results

5.1 f) i: Participants Twenty-two women with BC responded to recruitment letters, and after further screening, six

were excluded due to a history of diabetes (n=2), MRI incompatibility (n=2), and failure to

respond to follow-up calls regarding scheduling (n=2). Of the remaining sixteen BC survivors

who enrolled, fifteen received epirubicin, one received doxorubicin, fifteen received radiation

therapy, and none received Trastuzumab (Table 1). On average, BC survivors were studied

12.84.7 months after administration of the final anthracycline treatment. The groups were well-

matched for age, weight, body mass index, hemoglobin, cardiovascular risk factors, and self-

reported exercise (Table 1).

5.1 f) ii: Cardiorespiratory Fitness Peak cycle ergometer power output (132±31 vs. 165±30 W, p<0.01), absolute peak VO2

(1.69±0.37 vs. 2.13±0.41 L/min, p<0.01) and peak VO2 indexed to body mass (23.1±7.5 vs.

29.5±7.7 ml/kg/min, p=0.02) were significantly lower in BC survivors compared to controls.

Peak exercise heart rate, SaO2, respiratory exchange ratio, and rating of perceived exertion did

not differ between groups (Table 2).

Skeletal muscle oxidative capacity

Resting Pi:PCr ratio and pH were similar in both groups (Table 2). Peak PFE power

output, heart rate, rating of perceived exertion, Pi:PCr ratio and pH were not different between

groups (Table 2). Skeletal muscle oxidative capacity, measured as Pi:PCr ratio at a moderate-

intensity sub-maximal exercise workloads and by post-exercise PCr recovery τ did not differ

between BC and controls, with similar results for pH (Table 2)

109

5.1 f) iii: Rest and sub-maximal exercise lower leg VO2 and its determinants

Resting lower leg SvO2 was lower in the BC as compared to control group (745 vs

805%, p<0.01), corresponding to a higher O2 extraction ratio (0.240.06 vs 0.180.05, p=0.01),

while leg blood flow was lower in the BC group (5523 vs 7729 ml/min, p=0.02) resulting in

no difference between groups for resting leg VO2 (Table 2). Sub-maximal exercise power output,

VO2, blood flow, SvO2 and O2 extraction were similar between groups (Table 2, Figure 3).

Body composition

Subcutaneous fat volume did not differ between groups for the abdomen, thigh or lower leg

regions (Table 3). In the abdomen, visceral fat volume was higher (BC 406217 vs 273+123 mL,

p=0.04), paraspinal SM volume was lower (31339 vs 34032 ml, p=0.04), but absolute IMF

volume, IMF:SM, and SM fat fraction did not differ between groups. In the thigh, SM volume

did not differ between groups, but IMF volume (8231 vs 5817 ml, p=0.01), IMF:SM ratio

(0.360.19 vs 0.220.07, p=0.01) and SM fat fraction (2510 vs 185, p=0.02) were all

significantly higher in BC compared to controls. In the lower leg SM was reduced (752149 vs

903162 ml, p=0.01; Table 3), IMF volume did not differ, while the IMF:SM ratio (0.100.06 vs

0.060.02, p=0.03) and the SM fat fraction (95 vs 52%, p=0.02) (Table 3) were significantly

higher in BC.

5.1 f) iv: Relationship of skeletal muscle and intermuscular fat with whole-

body peak VO2 On analysis of all study participants combined, the IMF:SM ratio in the paraspinal, thigh,

and lower leg muscle groups each had a strong, inverse relationship to whole-body relative peak

VO2 (r=-0.70, p<0.001, r= -0.72, p<0.001, and r=-0.66, p<0.001 respectively; Figure 3). These

relationships were similarly strong in the BC group (paraspinal: r=-0.70, p=0.003; thigh: r= -

110

0.71, p=0.002; lower leg: r=-0.68, p=0.003). The SM fat fraction had a slightly stronger

relationship with whole-body peak VO2 than the IMF:SM ratio, in all regions (paraspinal: r=-

0.73, p<0.001; thigh: r= -0.78, p<0.001; lower leg: r=-0.71, p<0.001).

5.1 f) v: Relationship of lower leg muscle composition, resting and exercise

metabolism, blood flow, oxygen consumption, and extraction The lower leg IMF:SM ratio was not related to lower leg peak power output, Pi:PCr ratio, or

pH (Table 4). Lower leg IMF:SM ratio correlated strongly and positively with lower leg sub-

maximal exercise SvO2 (r=0.70, p<0.001). The IMF:SM ratio also had a strong inverse

relationship with lower leg sub-maximal exercise VO2 and O2 extraction (r=-0.64, p=0.008; r=-

0.67, p<0.001) and a moderate inverse relationship with lower leg exercise blood flow (r=-0.35,

p=0.05; Table 4). These relationships were similarly evident within the BC group alone, except

for blood flow, where the correlation effect size was similar but no longer significant due to

greater variability (r=-0.37, p=0.16).

Discussion

Peak VO2 is a strong independent predictor of CVD, disability and mortality in healthy

and clinical populations.149 Following BC treatment, women have a peak VO2 that is on average

20% lower than age-matched healthy sedentary women.13, 16, 17, 19, 124, 137 The mechanisms

responsible for the reduced exercise tolerance are not well understood, however recent evidence

suggests that ‘non-cardiac’ peripheral abnormalities associated with chemotherapy may be

important contributors to reduced exercise tolerance.29, 31 The major new finding of this study is

that despite a significantly lower peak VO2 during whole body exercise, BC survivors had

preserved lower leg blood flow, leg VO2 and mitochondrial oxidative capacity relative to

controls during unilateral PFE. A second novel finding is that the IMF:SM ratio and SM fat

111

fraction in the thigh and lower leg are strongly related to peak VO2, explaining approximately

50% of the variability in cardiorespiratory fitness. We also uncovered potential mediating

mechanisms for the impact of the IMF:SM ratio on local VO2. Specifically, a higher IMF:SM

ratio in the lower leg was associated with increased SvO2, reduced muscle VO2, blood flow, and

O2 extraction with moderate-intensity unilateral PFE. Taken together, these results suggest that

the primary peripheral impairments evident approximately one-year after anthracyclines are

abnormalities in skeletal muscle composition, namely increased IMF, which is an important

contributor to the reduced whole-body VO2 and O2 extraction in BC survivors.

5.1 f) vi: Skeletal mitochondrial oxidative capacity in BC survivors Contrary to our hypothesis, we did not observe impaired muscle function or reduced

oxidative capacity in our group of BC survivors. Specifically, in response to unilateral PFE for

which maximal blood flow is not limited by convective O2 delivery, the peak PFE workloads

achieved by the BC survivors did not differ from the controls. This was unexpected given the

significant and marked reduction in cycle ergometer power output achieved by the BC group

relative to the control group. Further, the Pi:PCr ratio, which reflects coupling of ATP use and

resynthesis, did not differ between groups at rest or at moderate and peak intensity exercise,

indicating similar patterns of aerobic and anaerobic metabolism during exercise (Table 2).

Notably, the peak pH at the end of exercise was similar between groups, indicating comparable

states of metabolic acidosis, allowing for comparison of post-exercise PCr recovery.150 The PCr

post-exercise recovery τ, a measure of mitochondrial ATP synthesis,151-153 was not different

between groups, nor blood flow, a potential confounding factor (Table 2). Taken together, these

results suggest that, on average, women who received anthracyclines for BC do not have

impaired SM mitochondrial function in response to small muscle mass exercise where the

limiting role of the heart is minimized.

112

Using vastus lateralis skeletal muscle biopsy, Mijwel et al. showed that during receipt of

anthracycline chemotherapy, women with BC experienced a reduction in oxidative muscle

fibers, citrate synthase, and capillarity.31 These deteriorations were attributed to deconditioning

rather than a result of chemotherapy as a 16-week exercise intervention consisting of high-

intensity aerobic and resistance interval training during adjuvant chemotherapy attenuated the

decline in mitochondrial function.31 In the present study, the women with BC were on average 13

months post-anthracycline treatment, and 75% self-reported performing some physical activity in

the previous month. It is possible that this length of time since treatment and/or the physical

activity performed may have contributed to recovery or preservation of mitochondrial function.

5.1 f) vii: Skeletal Muscle Composition and its relationship to VO2 Haykowsky et al. previously reported that the reduced peak VO2 in patients with heart failure

and preserved ejection fraction (HFpEF) was associated with a higher thigh IMF:SM ratio.30

Similarly, Reding et al. found inverse correlations between peak VO2 and paraspinal IMF:SM ratio

in 14 cancer survivors with mixed diagnoses and timing since treatment.29 However, Reding et al.

did not find differences in absolute amounts of IMF or the IMF:SM ratio of the paraspinal muscles

between cancer survivors and non-cancer controls. In our study, we used a similar approach to

assess the paraspinal muscle and found a similarly strong relationship between IMF:SM ratio and

whole-body peak VO2. We also assessed composition of the thigh and lower leg muscles, as the

primary muscles driving oxygen uptake during cycle and PFE. We extended prior non-cancer and

cancer studies by demonstrating that BC survivors previously treated with anthracycline

chemotherapy have increased thigh IMF, IMF:SM ratio, SM fat fraction, and reduced lower leg

SM, and increased lower leg IMF:SM and SM fat fraction compared to controls. Moreover, while

the average IMF:SM ratio and SM fraction varied among the paraspinal, thigh, and lower leg

muscles, it was inversely related to peak whole-body VO2 during cycle exercise in all three regions,

113

implicating IMF in the pathophysiology of anthracycline-related VO2 deterioration. A further

illustration of the extent of impairment in SM composition among women with BC is that the

IMF:SM ratio in our BC group was similar to that reported by Haykowsky et al. in patients with

HFpEF.30

We have further added to the understanding of the impact of IMF on SM metabolism by using

novel MRI techniques to non-invasively assess lower leg local VO2 and O2 extraction during

submaximal PFE. We did not find any relationship between IMF:SM and SM exercise metabolism

as assessed by 31P spectroscopy. However, lower leg IMF:SM ratio was inversely related to local

exercise VO2 and O2 extraction in both groups (Table 4). Specifically, the greater the amount of

IMF relative to the amount of SM, the less O2 consumed or extracted with exercise. That we found

this relationship between IMF:SM and VO2 for both whole-body and small muscle mass exercise

indicates that the impact of IMF on VO2 is independent of potential ‘central’ limitations. A

potential explanation for this IMF effect can be drawn from a study demonstrating that blood flow

to subcutaneous fat adjacent to exercising muscle increases in an intensity-dependent manner.131

This study of subcutaneous fat proposed potential mechanisms of vasodilation of adipose tissue

vessels including: 1) heat conduction from working muscle; 2) adenosine formation that occurs in

fat during exercise; and 3) conductive vasodilation up the arterial tree. It is possible that a similar

effect occurs in IMF where blood flow is shunted through the metabolically inactive IMF, resulting

in increased mixed venous O2 content.131 Consequently, this could explain the strong relationship

between lower leg IMF:SM ratio and both SvO2 and O2 extraction (Table 4). Another potential

mechanism for the impact of IMF on oxygen extraction is that the deposits of adipose tissue in the

muscle could reduce muscle oxygen diffusive conductance due to a greater distance for O2 to travel

from the capillaries to muscle mitochondria and a limited ability to locally restrict vascular

114

conductance in adipose tissue in close proximity to active SM.30 Finally, others have suggested

that IMF may result in lower overall blood flow to a tissue154; indeed we observed a negative

correlation between exercise blood flow to the entire lower leg (i.e., both fat and lean tissue) and

IMF:SM in both groups combined (Table 4).

In other populations, increased levels of IMF are associated with inflammation, insulin

resistance, reduced muscular activation, and reduced physical function.133 In combination with the

findings from the current study, prevention and reduction of IMF accumulation should be an

important goal for individuals with cancer. Both exercise and dietary interventions can reduce

IMF, but in either case, it appears that weight loss is necessary to achieve reductions in IMF.133

Weight loss induced by exercise may more effectively reduce IMF compared to weight loss

induced by caloric restriction.134-136, 155 Furthermore, the combination of caloric restriction and

aerobic exercise appears to be substantially more effective for reducing IMF compared to caloric

restriction and resistance exercise in premenopausal obese women.155

Limitations

A limitation of this study is that lower leg VO2 and its determinants were only measured

at rest and sub-maximal PFE. While caution is warranted in extending these measures to

maximal PFE, it is unlikely that group differences would exist, as the Pi:PCr ratio and

intracellular pH during and after incremental to maximal exercise were not different between

groups. Finally, blood flow was measured in the superficial femoral vein and was assumed to be

representative of lower leg venous return. Future studies may consider using positron emission

tomography or arterial spin labeling techniques to measure skeletal muscle perfusion during both

isolated and whole-body exercise.

115

5.1 g) Conclusion In women with BC treated with anthracycline chemotherapy one year earlier, peak VO2

and power output during whole body (cycle) exercise are significantly reduced relative to age-

and BMI-matched controls. However, during small muscle mass exercise that is not limited by

cardiac reserve, women with BC were able to perform a similar amount of work with a similar

lower leg VO2, blood flow, O2 extraction and oxidative capacity to controls. The primary

peripheral anomaly in BC compared to controls was in SM composition, with an increased

IMF:SM ratio and SM fat fraction in the thigh and lower leg among the women with BC. This is

an importance difference as a higher IMF:SM ratio was associated with lower whole-body peak

VO2 and reduced lower leg blood flow, VO2 and O2 extraction. Therefore, IMF appears to play

an important role in the impaired cardiorespiratory fitness reported following completion of BC

treatment. Diet and exercise interventions targeted to reduce the relative amount of IMF and

increase the absolute amount of SM in the legs should be investigated as possible therapies to

improve cardiorespiratory fitness following cancer therapy.

116

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5.1 a) Figure Legends:

5.1 a) i: Figure 1: A) Experimental set up for plantarflexion exercise device. B) Image acquisition for

determination of lower-leg VO2, showing the typical location for imaging of the superficial

femoral vein and representative SvO2 and blood velocity images, at rest and immediately post-

exercise. In this subject, SvO2 ≈ 86% at rest and 57% post exercise, with corresponding blood

velocities of 7 cm/s and 25 cm/s.

5.1 a) ii: Figure 2: MR body composition acquisition and analysis by the modified Dixon fat-water separation

approach in the A) lower leg, B) thigh, and C) abdomen of a representative participant with low

intermuscular fat and in the D) lower leg, E) thigh and F) abdomen of a participant with high

intermuscular fat.

5.1 a) iii: Figure 3: Relationship between IMF: SM ratio and whole-body peak VO2 for the paraspinal, thigh and

lower leg muscle regions.

120

5.1 b) Figures:

5.1 b) i: Figure 1: A) Experimental set up for plantarflexion exercise device. B) Image

acquisition for determination of lower-leg VO2, showing the typical location for imaging of the

superficial femoral vein and representative SvO2 and blood velocity images, at rest and

immediately post-exercise. In this subject, SvO2 ≈ 86% at rest and 57% post exercise, with

corresponding blood velocities of 7 cm/s and 25 cm/s.

Figure 9 Experimental set-up for plantarflexion exercise and image acquisition for determination of lower-leg oxygen uptake

121

5.1 b) ii: Figure 2: MR body composition acquisition and analysis by the modified Dixon

fat-water separation approach in the A) lower leg, B) thigh, and C) abdomen of a representative

participant with low intermuscular fat and in the D) lower leg, E) thigh and F) abdomen of a

participant with high intermuscular fat.

Figure 10 MRI body composition acquisition and analysis

122

5.1 b) iii: Figure 3: Relationship between IMF: SM ratio and whole-body peak

VO2 for the paraspinal, thigh and lower leg muscle regions. Figure 11 Relationship between IMF:SM ratio and whole body peak oxygen uptake

123

5.1 c) Tables:

5.1 c) i: Table 1: Participant characteristics. Table 9 Participant characteristics

Parameter BC

(n=16)

Control

(n=16)

P

value

Age (years) 56 (10) 56 (10) 0.95

Weight (kg) 75.6 (12.0) 74.9 (11.7) 0.87

Body Mass Index (kg/m2) 29.0 (3.6) 27.9 (4.9) 0.47

Self-Reported Exercise

(minutes/week)*

225 (275) 313 (208) 0.16

Hemoglobin (g/L) 133 (11) 134 (8) 0.67

Hematocrit (%) 40 (3) 41 (2) 0.51

Cardiovascular Risk Factors

Sedentary (%) 25% 13% 0.65

Hypertension (%) 13% 0% 0.48

Hypercholesterolemia (%) 0% 13% 0.48

Former Smoker (%) 44% 50% 1

Current Smoker (%) 0% 0% 1

Diabetes (%) 0% 0% 1

Overweight (%) 31% 50% 0.47

Obese (%) 44% 44% 1

Other Comorbid Conditions

Arthritis (%) 31% 44% 0.72

Osteoporosis (%) 13% 6% 1

Thyroid Disorder (%) 13% 19% 1

Lung Disease (%) 0% 0% 1

Breast Cancer Characteristics

Stage II/III/IV (n) 8/7/1 - -

Epirubicin/Doxorubicin (n) 15/1 - -

Dose (mg/m2) 307 (75) - -

Does Reduction (n) 3 - -

No Surgery/ Lumpectomy/

Mastectomy (n)

1/6/9 - -

Radiation Therapy (n) 15 - -

Hormone Therapy (n) 11 - -

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5.1 c) ii: Table 2: Cardiopulmonary exercise performance during cycle

exercise and lower leg muscle metabolism and oxygenation at rest and after

plantar flexion exercise in BC survivors and controls. Table 10 Cardiopulmonary exercise performance and lower leg muscle bioenergetics

Parameter BC Control p value

Peak Cycle Ergometry

Power output (W) 132 (31) 165 (30) <0.01

Oxygen uptake (l/min) 1.69 (0.37) 2.13 (0.41) <0.01

Oxygen uptake (ml/kg/min) 23.1 (7.5) 29.5 (7.7) 0.02

Respiratory exchange ratio 1.29 (0.09) 1.27 (0.08) 0.60

SaO2 (%) 96 (2) 94 (2) 0.08

Heart rate (bpm) 166 (12) 161 (14) 0.35

Rating of perceived exertion (0-10

scale)

9 (1) 9 (1) 0.68

Rest (lower leg)

Pi:PCr 0.10 (0.03) 0.11 (0.03) 0.24

pH 7.06 (0.06) 7.08 (0.10) 0.44

Blood flow (ml/min) 55 (23) 77 (29) 0.02

Pulse oximeter SaO2 (%) 97 (2) 98 (2) 0.36

SVO2 (%) 74 (5) 80 (5) <0.01

O2 extraction ratio 0.240.06 0.180.05 0.01

VO2 (ml/min) 2.1 (0.84) 2.3 (0.54) 0.64

Sub-maximal (60% of peak) plantar flexion exercise

Power output (W) 10.9 (1.2) 12.1 (2.4) 0.09

Peak blood flow (ml/min) 526 (171) 581 (198) 0.41

Peak SvO2 (%) 55 (7) 54 (5) 0.81

Peak O2 extraction ratio 0.440.07 0.450.06 0.72

125

Peak VO2 (ml/min) 40 (15) 45 (15) 0.31

Peak plantar flexion exercise

Power output (W) 18.0 (2.4) 19.6 (3.4) 0.13

Heart rate (bpm) 102 (16) 100 (20) 0.84

Rating of perceived exertion (0-10

for lower leg)

10 (1) 10 (0) 0.28

Pi:PCr 0.98 (0.32) 0.90 (0.24) 0.41

pH 6.46 (0.22) 6.45 (0.28) 0.39

Recovery (lower leg)

PCr τ (s) 36 (11) 33 (9) 0.33

126

5.1 c) iii: Table 3: Body composition in the thigh, lower leg and abdomen.

*Thigh data is for 4 cm of transverse slices starting at 4 cm from distal end of

femur. †Lower leg data is for whole lower leg starting 5 cm below tibial plateau

until distal end of gastrocnemius.‡ Lumbar spine data is 1.2 cm section of

transverse images at the center of the third lumbar vertebrae.

Table 11 Body

composition of the thigh, lower leg and abdomen

Variable BC (n=16) Controls (n=16) P-value

Thigh* Total volume (mL) 75483 771147 0.70

Subcutaneous fat (g) 39783 420127 0.55

Muscle (g) 24953 26637 0.27

Intermuscular fat (g) 8231 5817 0.01

IMF:SM 0.360.19 0.220.07 0.01

SM fat fraction (%) 2510 185 0.02

Lower leg†

Total volume (mL) 1599285 1884395 0.03

Subcutaneous fat (g) 659189 787264 0.13

Muscle (g) 752149 903162 0.01

Intermuscular fat (g) 6532 5123 0.06

IMF:SM 0.100.06 0.060.02 0.03

SM fat fraction (%) 95 52 0.02

Abdomen‡

Total volume 1684471 1490473 0.25

Subcutaneous fat 897258 823379 0.52

Muscle 31339 34032 0.04

Intermuscular fat 6927 5328 0.13

IMF:SM 0.220.10 0.160.09 0.06

SM fat fraction 186 136 0.06

Visceral fat 406217 273+123 0.04

127

5.1 c) iv: Table 4: Pearson correlations between lower leg IMF:SM ratio and

skeletal muscle function, resting and exercise metabolism. Table 12 Pearson's correlations between lower leg IMF:SM ratio and skeletal muscle bioenergetics

BC group

(n=16)

All participants

(n=32)

r p-value r p-value

Peak plantar flexion power output -0.24 0.38 -0.29 0.11

Peak plantar flexion Pi:PCr -0.20 0.46 -0.17 0.35

Peak plantar flexion pH 0.42 0.11 0.22 0.21

Pi:PCr @ ~60% peak power 0.21 0.44 0.07 0.71

pH @ ~60% peak power 0.12 0.66 0.17 0.35

PCr recovery tau 0.03 0.92 0.14 0.43

Lower leg exercise blood flow -0.37 0.16 -0.35 0.05

Lower leg exercise VO2 -0.63 0.008 -0.64 0.008

Lower leg exercise SvO2 (%) 0.76 <0.001 0.70 <0.001

Lower leg exercise O2 extraction -0.71 0.002 -0.67 <0.001

128

6.1 Conclusion

129

Breast cancer (BC) mortality has decreased by more than 40% over the last three

decades.122 Accordingly, cardiovascular health is becoming an increasingly important focus

when planning cancer treatment, and care beyond antineoplastic therapy. Anthracyclines are

proven to provide a BC survivorship advantage, however, at the potential cost of cardiovascular

health.6 We developed and used non-invasive imaging technology to comprehensively

investigate pathophysiologic mechanisms leading to cardiovascular decline in BC survivorship.

The strongest predictor of all-cause and cardiovascular mortality in healthy and clinical

populations is VO2 peak, which is reduced by 20% in BC survivors relative to age- and sex-

matched control subjects. In Chapter 3, we demonstrate reduced VO2 peak in BC patients prior to

receipt of anthracycline chemotherapy. In these patients, using state of the art exercise cMRI we

found that peak cardiac output is reduced relative to controls. Importantly, the lower exercise

cardiac output was the result of reduced cardiac size rather than cardiac dysfunction, and was

evident in the absence of a cardiotoxic stimulus. Supine peak cardiac output was significantly

related to upright VO2 peak, but explained less than half of the variance in effect.

We further investigated VO2 peak deficits in BC survivors by studying non-cardiac,

peripheral determinants in Chapter 4. Using single leg knee extension exercise, where the

limiting role of the heart is minimized, we found that long-term BC survivors (12 years post

anthracycline therapy) had no decrement in peak work rate or leg blood flow, but had reduced

estimated quadriceps mass. The finding of preserved leg blood flow in the face of reduced

muscle mass implicates uncoupling of the former with oxygen use (i.e. inefficient delivery of

blood to active muscle). In Chapter 5, using novel MR strategies, we further investigated this

phenomenon to comprehensively measure leg composition, lower leg blood flow, and muscle

oxidative capacity. Specifically, BC survivors (>3 months post anthracycline therapy) and age-

130

and sex matched controls underwent incremental to maximal plantar flexion exercise, another

paradigm whereby the limiting role of the heart is minimized. Peak work rate, peak Pi:PCr ratio,

muscle acidity, and mitochondrial oxidative capacity in the lower leg as measured by 31PMRS

were not different between BC survivors and controls. Further, in response to submaximal

plantarflexion exercise, no differences were found in lower-leg blood flow, oxygen extraction or

VO2, despite a severe reduction in whole-body VO2 peak. However, BC survivors had poor body

composition relative to controls, as indicated by reduced muscle mass, increased adiposity, or an

unfavorable shift in the ratio of adipose tissue to skeletal muscle. Whole body VO2 peak was

strongly related to muscle composition; specifically, an inverse relationship was found between

the intermuscular fat (IMF) to skeletal muscle (SM) ratio and VO2 peak. An increased IMF:SM

ratio could impair VO2 peak through mechanisms including: 1) shunting of blood through IMF,

resulting in poor matching of blood flow to metabolic demand; 2) diffusive conductance

limitations resulting in impaired oxygen extraction; or 3) insulin resistance mediated

perturbations in oxidative SM function resulting in reduced oxygen utilization. The relationship

between IMF:SM ratio and VO2 peak was evident across BC and controls; BC had a greater

IMF:SM ratio in the thigh, the predominant source of oxygen consumption during

cardiopulmonary (cycle) exercise testing.

Taken together, our findings do not support a causal relationship between anthracycline

toxicity and exercise intolerance. Several studies report underlying VO2 peak deficits in BC prior

to receipt of anthracycline therapy, and we demonstrate that reductions in peak cardiac output are

present prior to anthracycline administration (Chapter 3).109, 124 Further, our findings do not

support unique peripheral mechanisms of anthracycline related exercise intolerance. Instead,

reduced VO2 peak was strongly linked to skeletal muscle mass and muscle composition, with a

131

propensity of BC survivors having higher adiposity and lower muscle mass relative to disease

and treatment naive control subjects. Importantly, neither this phenotype, nor the

cardiopulmonary effects of this phenotype are unique to BC (Chapter 5).

While we observed reduced VO2 peak prior to administration of anthracycline therapy,

there is convincing evidence of further reductions in VO2 peak from pre-to-post-therapy. Early

evidence is available to establish a working theory of physiologic mechanisms leading to

reduced VO2 peak in BC. First, Howden et al. reported that VO2 peak declined by 15% from pre (0

months) to post anthracycline therapy (4 months) in BC patients (n=14).156 Further, this decline

was attributed to a reduction in calculated arterial-venous oxygen content difference (a-vO2), as

peak cardiac output was unchanged by anthracycline therapy. In the same study, a group of BC

patients (n=14) underwent an exercise training intervention which maintained VO2 peak, by

preserving peak cardiac output and a-vO2 difference. Moreover, a subset of participants who

were followed up at 16 months, at which time changes in VO2 peak persisted from 4 months, but

did not worsen.123 By 16 months, peak cardiac output was significantly reduced as a result of

reduced biventricular ejection fraction and ejection fraction reserve. Notably, this change in peak

cardiac output indicates a “compensatory” increase in a-vO2 difference to preserve VO2 peak. One

interpretation of these findings is that aberrant peripheral changes occur, resulting in later cardiac

adaptation towards normal cardiac output to VO2 peak matching.

In support of this theory, one study is available providing information on muscle

composition and quality changes from pre-to-post-anthracycline therapy. Mijwel et al. provide

strong evidence of muscle deterioration using baseline and post-therapy vastus lateralis biopsy.31

In the usual care group, mitochondrial oxidative capacity and type I (oxidative) muscle fiber

cross-sectional area decreased. In the same study, intervention groups underwent resistance or

132

aerobic exercise training which preserved mitochondrial function and muscle fiber composition.

The authors proposed that changes observed in the usual care group were related to anthracycline

associated reductions in physical activity, rather than anthracycline myo-toxicity. Indeed, these

results align well with a study from nearly 2-decades earlier that found that adjuvant therapy was

associated with a reduction in physical activity without an accompanying reduction in caloric

intake, leading to muscle loss with net weight gain.27 The consequence of reduced muscle mass

and increased adiposity is reduced VO2 peak.

Taken together with our finding of a physiologic link between an increased IMF:SM ratio

and reduced VO2 peak, it is possible that peak cardiac output adapts to the minimal value needed

to meet peripheral demands. Indeed, we observed increasing VO2 peak with increasing cardiac

output (Chapter 3) as well as decreasing IMF:SM ratio (Chapter 5) independent of a history of

BC. Accordingly, preserving or improving body composition should be an important goal and

clinical endpoint when designing interventions to improve cardiovascular function in BC

survivorship. Critically, there is already a wealth of data demonstrating the efficacy of exercise

training to preserve or improve muscle composition in older women, while cardiac adaptation

presents a greater challenge.157-159 Data from Foulkes et al. demonstrating aberrant cardiac

changes only 12 months after completing therapy stresses the importance of initiating cardio-

protective and/or body composition protective exercise training interventions during, or

immediately after completion of adjuvant therapy.123 To this end, future studies are required to

test effectiveness of interventions at increasing VO2 peak and curving CV risk. In consideration of

clinical feasibility of an exercise intervention, strategies should consider the cost effectiveness,

the minimal amount of exercise needed to preserve cardiorespiratory fitness, patient feasibility,

and long term sustainability.

133

6.1 a) References 6. Kirkham AA, Beaudry RI, Paterson DI, Mackey JR and Haykowsky MJ. Curing breast cancer

and killing the heart: A novel model to explain elevated cardiovascular disease and mortality risk among

women with early stage breast cancer. Prog Cardiovasc Dis. 2019.

27. Demark-Wahnefried W, Peterson BL, Winer EP, Marks L, Aziz N, Marcom PK, Blackwell K and

Rimer BK. Changes in weight, body composition, and factors influencing energy balance among

premenopausal breast cancer patients receiving adjuvant chemotherapy. Journal of clinical oncology :

official journal of the American Society of Clinical Oncology. 2001;19:2381-9.

31. Mijwel S, Cardinale DA, Norrbom J, Chapman M, Ivarsson N, Wengstrom Y, Sundberg CJ and

Rundqvist H. Exercise training during chemotherapy preserves skeletal muscle fiber area, capillarization,

and mitochondrial content in patients with breast cancer. FASEB journal : official publication of the

Federation of American Societies for Experimental Biology. 2018;32:5495-5505.

109. Peel AB, Thomas SM, Dittus K, Jones LW and Lakoski SG. Cardiorespiratory Fitness in Breast

Cancer Patients: A Call for Normative Values. JOURNAL OF THE AMERICAN HEART ASSOCIATION.

2014;3:e000432.

122. Siegel RL, Miller KD and Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019;69:7-34.

123. Foulkes S, Howden EJ, Bigaran A, Janssens K, Antill Y, Loi S, Claus P, Haykowsky MJ, Daly

RM, Fraser SF and La Gerche A. Persistent Impairment in Cardiopulmonary Fitness following Breast

Cancer Chemotherapy. Med Sci Sports Exerc. 2019.

124. Beaudry R, Howden, E., Foulkes, S., Bigaran, A., Claus, P., Haykowsky, M., La Gerche, A.

Determinants of Exercise Intolerance in Breast Cancer Patients Prior to Anthracycline Chemotherapy.

Physiological Reports. 2019;7:e13971.

156. Howden E, Bigaran, A., Beaudry, R., Fraser, S., Selig, S., Foulkes, S., Antil, A., Nightingale, S.,

Loi, S., Haykowsky, M., La Gerche, A. Exercise as a diagnostic and therapeutic tool for the prevention of

functional disability in breast cancer patients. European Journal of Preventive Cardiology (Accepted).

2018.

157. Hersey WC, 3rd, Graves JE, Pollock ML, Gingerich R, Shireman RB, Heath GW, Spierto F,

McCole SD and Hagberg JM. Endurance exercise training improves body composition and plasma insulin

responses in 70- to 79-year-old men and women. Metabolism. 1994;43:847-54.

158. Peterson MD, Sen A and Gordon PM. Influence of resistance exercise on lean body mass in aging

adults: a meta-analysis. Medicine and science in sports and exercise. 2011;43:249-258.

159. Howden EJ, Sarma S, Lawley JS, Opondo M, Cornwell W, Stoller D, Urey MA, Adams-Huet B

and Levine BD. Reversing the Cardiac Effects of Sedentary Aging in Middle Age—A Randomized

Controlled Trial: Implications For Heart Failure Prevention. Circulation. 2018;137:1549-1549.

134

7.1 Appendix

135

7.1 a) Chapter 3 Data Supplement

0

25

50

75

Age

(Ye

ars)

1

1.5

2

2.5B

od

y Su

rfac

e A

rea

(m²)

15

25

35

45

Bo

dy

Mas

s In

dex

(Kg

/m²)

Supplemental Figure 1:

Distributions of participant characteristics for A)

age, B) body mass index and C) body surface area.

No differences were found between BC subgroups,

or BC subgroups and healthy controls.

A

B

C

136

10

20

30

40

Peak

VO

2

(ml/

kg/m

in)

1

2

3

Peak

VO

2 (l

/min

)

45

60

75

90

105

LV E

DV

(m

l/m²)

9

12

15

18

21

Peak

Car

dia

c O

utp

ut

(l/m

in)

10

20

30

40

Pre

dic

ted

VO

2

(ml/

kg/m

in)

Supplemental Figure 2:

Distributions of A) predicted peak VO2, B) absolute

peak VO2 C) relative VO2 D) left ventricular end-

diastolic volume and E) peak cardiac output No

differences were found between BC subgroups. Both

subgroups were significantly different (p<0.01)

versus controls for all variables.

A

B C

D E

137

Supplemental Table 1:

Mean, standard deviation and p values (student’s t-test) for breast cancer subgroups versus healthy

controls. Significant values are bolded.

Characteristic Neoadjuvant

Subgroup

Adjuvant

Subgroup

Healthy

Control

P; Neoadjuvant

vs Adjuvant

P; Neoadjuvant vs

Healthy Control

P; Adjuvant vs

Healthy Control

Age

46 (14) 49 (9) 48 (12) 0.56 0.80 0.79

BMI

25.8 (6) 28 (8) 24 (2) 0.51 0.33 0.16

BSA

1.84 (0.2) 1.72 (0.2) 1.73 (0.1) 0.13 0.15 0.92

VO2

(ml/kg/min)

25 (4) 25(8) 35 (6) 0.94 0.00009 0.002

VO2 (l/min)

1.77 (0.3) 1.68 (0.4) 2.29 (0.5) 0.52 0.007 0.004

Peak Power

(Watts)

146 (39) 134 (51) 228 (51) 0.50 0.0003 0.0002

Peak Cardiac

Output (l/min)

13.7 (1.3) 13.5 (2.1) 17.0 (2.2) 0.75 0.0005 0.0008